CN108884147B - Fibronectin-based scaffold molecules that bind glypican 3 - Google Patents

Abstract

Provided herein are polypeptides comprising the tenth domain of fibronectin type III ( ^10Fn3 ) that bind to glypican 3. Fusion molecules containing a ¹⁰ Fn3 domain that binds glypican 3 for use in diagnostic and therapeutic applications are also provided. Glypican ^3-10 Fn3 drug conjugates are also provided.

Description

Fibronectin-based scaffold molecules that bind glypican 3

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 62/222,633 filed on September 23, 2015, the contents of which are expressly incorporated herein by reference. The contents of any patents, patent applications, and references cited throughout this specification are fully incorporated herein by reference.

background

Glypican 3 is a carcinoembryonic antigen belonging to the glypican family, which is composed of heparan sulfate proteoglycans anchored by glycosylphosphatidylinositol. Glypican regulates the activity of several growth factors, including Wnts, Hedgehogs, bone morphogenetic protein and fibroblast growth factor (FGF) (Filmus et al., FEBS J. 2013, 280: 2471-2476). Glypican is characterized by covalent attachment to a polysaccharide chain called heparan sulfate glycosaminoglycan. Glypican participates in cell signal transduction at the cell-extracellular matrix interface. (Sasisekharan et al., Nature Reviews|Cancer, Volume 2 (2002). To date, six different members of the human glypican family have been identified. Cell membrane-bound glypican 3 consists of two subunits linked by one or more disulfide bonds.

Glypican 3 is expressed in the fetal liver and placenta during development and is downregulated or silenced in normal adult tissues. Mutations and deletions in the Glypican 3 gene are responsible for Simpson-Golabi-Behmel or Simpson malformation syndrome in humans. Glypican 3 is expressed in various cancers, particularly hepatocellular carcinoma ("HCC"), melanoma, Wilms' tumor, and hepatoblastoma. (Jakubovic and Jothy; Ex. Mol. Path. 82: 184-189 (2007); Nakatsura and Nishimura, Biodrugs 19 (2): 71-77 (2005). The cell surface form of Glypican 3 is highly expressed in HCC (>50%) and other cancers including squamous lung cancer (about 25%).

Glypican 3 promotes tumor growth in vitro and in vivo by stimulating canonical Wnt signaling, which induces cytoplasmic accumulation and nuclear translocation of the transcription factor β-catenin (Filmus, supra). It has been shown that GPC3 can form complexes with several Wnts (Capurro et al., Cancer Res., 2005, 65: 6245-6254) and Frizzleds (signaling receptors for Wnts) (Filmus et al., Genome Biol., 2008, 9: 224).

HCC is the third leading cause of cancer-related death worldwide. HCC causes approximately 1 million deaths each year. (Nakatsura and Nishimura, Biodrugs 19(2):71-77(2005).) Hepatitis B virus, hepatitis C virus, and long-term heavy drinking, which lead to cirrhosis, remain the most common causes of HCC. Due to the spread of hepatitis C virus infection, its incidence has increased dramatically in the United States and is expected to increase in the next 20 years. HCC is mainly treated by liver transplantation or tumor resection. The patient's prognosis depends on both the baseline liver function and the stage at which the tumor is diagnosed. (Parikh and Hyman, Am J. Med. 120(3):194-202(2007).) Effective HCC treatment strategies are needed.

Overview

Provided herein are polypeptides containing a fibronectin-based scaffold (FBS) that binds human glypican 3, and conjugates of these polypeptides that are suitable as therapeutic and diagnostic agents.

In one aspect, a polypeptide comprising a FBS that specifically binds to human glypican 3 (GPC3) is provided. In some embodiments, the anti-GPC3 FBS is conjugated to a therapeutic or diagnostic agent.

In another aspect, a method of treating cancer in a human subject by administering to the subject a therapeutically effective amount of a polypeptide comprising anti-GPC3FBS or an anti-GPC3FBS-drug conjugate is provided. In some embodiments, the cancer overexpresses Glypican 3 associated with non-cancerous cells. In some embodiments, the cancer is liver cancer (e.g., hepatocellular carcinoma, hepatoblastoma), melanoma, Wilms' tumor, or lung cancer (e.g., squamous cell lung cancer).

In another aspect, methods for detecting GPC3 in vitro and in vivo are provided, comprising contacting a cell with a polypeptide comprising an anti-GPC3 FBS under conditions that allow the anti-GPC3 FBS to bind to GPC3, and detecting a complex comprising the anti-GPC3 FBS and GPC3. In some embodiments, the anti-GPC3 FBS is linked to a detectable label (e.g., FITC).

Also provided are compositions comprising the anti-GPC3FBS polypeptides and/or anti-GPC3FBS-drug conjugates, including pharmaceutical compositions and diagnostic compositions.

Also provided are nucleic acid molecules encoding anti-GPC3FBS, expression vectors comprising such nucleic acids, and host cells comprising such expression vectors. Also provided are kits comprising anti-GPC3FBS polypeptides and anti-GPC3FBS conjugates and instructions for use.

Other characteristics and advantages of the invention will become apparent upon reading the following detailed description and examples, which are not to be interpreted as limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts the amino acid sequences of representative anti-GPC3 Adnectin polypeptides. 4578F03 (SEQ ID NO: 9), 4578H08 (SEQ ID NO: 18), 4578B06 (SEQ ID NO: 35), 4606F06 (SEQ ID NO: 48), 5273C01 (SEQ ID NO: 61), 5273D01 (SEQ ID NO: 74), 5274E01 (SEQ ID NO: 87), 6561A01 (SEQ ID NO: 87), 6077F02 (SEQ ID NO: 102), 6093A01 (SEQ ID NO: 463).

2A-2B are schematic diagrams depicting the structures of DAR1 and DAR2 tubulysin analog-GPC3 Adnectin drug conjugates.

Figures 3A-3D show flow cytometry results of anti-GPC3 Adnectin binding to human Glypican 3-positive cells.

Figures 4A-4B show flow cytometry results of anti-GPC3 AdxDC DAR1 and DAR2 antibody binding to human Hep3B and H446 cells.

5A-5B show cell growth inhibition of Hep3B and H446 cells by anti-GPC3 AdxDC DAR1 and DAR2 antibodies.

6A-6B show cell growth inhibition of Hep3B and HepG2 cells by anti-GPC3 AdxDC DAR1 and DAR2 antibodies.

7A-7B are bar graphs depicting the internalization rate of the anti-GPC3 Adnectin, ADX_6077_F02, into H446 and HepB3 cells.

Figures 8A-8F depict membrane and internalized AF488-labeled anti-GPC3 Adnectin, ADX_6077_F02, at time points 15 minutes and 8 hours.

FIG. 9 is a graph depicting the exposure profile of tubulysin analog-anti-GPC3 Adnectin drug conjugates in mice.

10A-10B are graphs depicting the effect of anti-GPC3 Adnectin drug conjugates in the HepG2 xenograft model as measured by tumor volume reduction and percent change in body weight.

11A-11D are graphs depicting the effects of DAR1 and DAR2 in the HepG2 xenograft model (TV ₀ =380-480 mm ³ ₎ as measured by tumor volume reduction and percent change in body weight.

12A-12B are graphs depicting the effect of DAR2 in the HepG2 xenograft model (TV ₀ =228-350 mm ³ ) as measured by tumor volume reduction and percent change in body weight.

13A-13B are graphs depicting the effect of DAR2 in the HepG2 xenograft model (TV ₀ =514-673 mm ³ ) as measured by tumor volume reduction and percent change in body weight.

FIG. 14 depicts the common peptic peptide of human GPC3 (amino acids 1-559 of SEQ ID NO: 344 followed by 6X his) determined by mass spectrometry.

FIG. 15 is a graphical depiction of the ADX_6077_F02 adnectin binding site on human GPC3 as determined by HDX-MS.

16A-16B are graphical comparisons of the binding kinetics of anti-GPC3 DG mutants and the parental anti-GPC3 Adnectin.

FIG. 17 shows tumor volume over days after administration of different doses of GPC3_AdxDC DA or control non-binding AdxDC to NSG mice implanted with Hep3B tumor cells, indicating that GPC3_AdxDC DA is effective in xenografts derived from cell lines that highly express Glypican 3.

FIG. 18 shows tumor volume over days after administration of different doses of GPC3_AdxDC DA or control non-binding AdxDC to CB17 SCID mice implanted with H446 tumor cells, indicating that GPC3_AdxDC DA delayed the growth of xenografts derived from a cell line that lowly expresses Glypican 3.

FIG. 19 shows quantitative whole body autoradiography (QWBA) of mouse tissues taken 0.17 hours, 1 hour, 5 hours, and 168 hours after administration of 0.22 μM/kg of ³ H GPC3_AdxDC to mice, demonstrating preferential uptake by Hep3B tumors relative to normal tissues.

FIG. 20 shows QWBA of mouse tissues taken 0.17 hours, 1 hour, 5 hours, and 168 hours after administration of 0.015 μM/kg of ³ H GPC3_AdxDC to mice, demonstrating preferential uptake by Hep3B tumors relative to normal tissues.

FIG. 21 shows QWBA of mouse tissues taken 0.17 hours, 1 hour, 5 hours, and 168 hours after administration of 0.22 μM/kg of ³ H GPC3_AdxDC to mice, demonstrating higher uptake of Hep3B tumors relative to non-binding control AdxDC.

FIG. 22 shows QWBA of mouse tissues taken 0.17 hours, 1 hour, 5 hours and 168 hours after administration of 0.22 μM/kg of ³ HRGE_AdxDC (control, non-GPC3 binding AdxDC) to mice, demonstrating lower uptake of Hep3B tumors relative to GPC3_AdxDC.

Figure 23 shows the total radioactivity concentration in different mouse tissues at 0.17 hours, 1 hour, 5 hours, 24 hours, 48 hours and 168 hours after administration of ^3H GPC3_AdxDC or non-binding AdxDC control ("RGE_ADxDC") to mice. The bars for each tissue are shown in the order stated in the previous sentence.

Figures 24A-24B show heat maps of the BC loop of GPC3_AdxDC DG (amino acids 15-21 of SEQ ID NO: 98) obtained by positional scanning of human Glypican 3-biotin at 10 nM (Figure 24A) or 1 nM (Figure 24B). The sequence of the wt BC loop is listed in amino acids 15-21 of SEQ ID NO: 1.

Figures 25A-25B show heat maps of the BC loop of GPC3_AdxDC DA (amino acids 15-21 of SEQ ID NO: 98) obtained by positional scanning of human Glypican 3-biotin at 10 nM (Figure 25A) or 1 nM (Figure 25B). The sequence of the wt BC loop is listed in amino acids 15-21 of SEQ ID NO: 1.

Figures 26A-26B show heat maps of the DE loop of GPC3_AdxDC DG obtained by positional scanning using 10 nM (Figure 26A) or 1 nM (Figure 26B) of human Glypican 3-biotin. The sequence of the wt DE loop is listed in amino acids 52-55 of SEQ ID NO:1.

Figures 27A-27B show heat maps of the DE loop of GPC3_AdxDC DA obtained by positional scanning using 10 nM (Figure 27A) or 1 nM (Figure 27B) of human Glypican 3-biotin. The sequence of the wt DE loop is listed in amino acids 52-55 of SEQ ID NO:1.

Figure 28 shows a heat map of the FG loop of GPC3_AdxDC DG (amino acids 69-79 of SEQ ID NO: 98) obtained by positional scanning using 10 nM human Glypican 3-biotin. The sequence of the wt FG loop is set forth in amino acids 77-87 of SEQ ID NO: 1.

Figure 29 shows a heat map of the FG loop of GPC3_AdxDC DG (amino acids 69-79 of SEQ ID NO: 98) obtained by positional scanning using 1 nM human Glypican 3-biotin. The sequence of the wt FG loop is set forth in amino acids 77-87 of SEQ ID NO: 1.

Figure 30 shows a heat map of the FG loop of GPC3_AdxDC DA (amino acids 69-79 of SEQ ID NO: 98) obtained by positional scanning using 10 nM human Glypican 3-biotin. The sequence of the wt FG loop is set forth in amino acids 77-87 of SEQ ID NO: 1.

Figure 31 shows a heat map of the FG loop of GPC3_AdxDC DA (amino acids 69-79 of SEQ ID NO: 98) obtained by positional scanning using 1 nM human Glypican 3-biotin. The sequence of the wt FG loop is set forth in amino acids 77-87 of SEQ ID NO: 1.

DETAILED DESCRIPTION OF THE INVENTION

definition

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled person. Although any methods and compositions similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and compositions are described herein.

The terms "glypican 3," "glypican proteoglycan 3," "GPC3," "OTTHUMP00000062492," "GTR2-2," "SGB," "DGSX," "SDYS," "SGBS," "OCI-5," and "SGBS1" are used interchangeably and include variants, isoforms, and species homologs of human glypican 3. The complete amino acid sequence of an exemplary human glypican 3 has a Genbank/NCBI accession number of NM_004484 (SEQ ID NO: 344).

An "amino acid residue" is the portion of an amino acid that remains after one water molecule (H+ from the nitrogen-containing side and OH- from the carboxyl side) has been lost in peptide bond formation.

By "polypeptide" is meant any sequence of two or more amino acids, regardless of length, post-translational modification, or function. Polypeptides may include natural and unnatural amino acids as those described in U.S. Pat. No. 6,559,126, which is incorporated herein by reference. Polypeptides may also be modified by any of a variety of standard chemical methods (e.g., amino acids may be modified with protecting groups; carboxyl terminal amino acids may be made into terminal amide groups; amino terminal residues may be modified with groups, e.g., to enhance lipophilicity; or, polypeptides may be chemically glycosylated or otherwise modified to increase stability or in vivo half-life). Polypeptide modifications may include the attachment of another structure, such as a cyclic compound or other molecule to the polypeptide, and may also include polypeptides containing one or more amino acids in an altered configuration (i.e., R or S; or, L or D).

An "isolated" polypeptide is one that has been identified and separated and/or recovered from components of its natural environment. Contaminating components of its natural environment are substances that would interfere with the diagnostic or therapeutic use of the polypeptide and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In certain embodiments, the polypeptide will be purified to: (1) greater than 95% by weight of the polypeptide as determined by the Lowry Method, and most preferably greater than 99% by weight, (2) to a degree sufficient to obtain at least the N-terminal residues or internal amino acid sequence by use of a spinning cup sequenator, or (3) to a degree of homogeneity by Coomassie Brilliant Blue or preferably silver staining of SDS-PAGE performed under reducing or non-reducing conditions.

As used herein, " ^10Fn3 domain" or " ^10Fn3 module" or " ^10Fn3 molecule" refers to wild- ^type10Fn3 and biologically active variants thereof, e.g., biologically active variants that specifically bind to a target, such as a target protein. The wild-type ^human10Fn3 domain may comprise the amino acid sequence set forth in SEQ ID NO: 1. Biologically active variants of the wild-type ^human10Fn3 domain include10Fn3 domains that comprise at least, at most, or about 1, ² , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, or 45 amino acid changes (i.e., substitutions, additions, or deletions) relative to ^the10Fn3 domain comprising SEQ ID NO: 1.

"Adnectin" or "Adx" or "adnectin" or "adx" refers to a ^human10Fn3 molecule that has been modified (relative to the wild-type sequence) to specifically bind to a target.

A "GPC3 Adnectin" or "anti-GPC3 Adnectin" is an Adnectin that specifically binds to GPC3, e.g., with a _KD of 1 μM or less.

As used herein, a "region" of ^a10Fn3 domain (or module or molecule) refers to the loops (AB, BC, CD, DE, EF and FG), the beta strands (A, B, C, D, E, F and G), the N-terminus (corresponding to amino acid residues 1-7 of SEQ ID NO: 1), or the C-terminus (corresponding to amino acid residues 93-94 of SEQ ID NO: 1).

The "North Pole loop" of ^a10Fn3 domain (or module) refers to any of the BC, DE, and FG loops of ^the10Fn3 domain.

The "South Pole loop" of ^a10Fn3 domain (or module) refers to any of the AB, CD, and EF loops of ^the10Fn3 domain.

"Scaffold region" refers to any non-loop region of the ^human10Fn3 domain. The scaffold region includes the A, B, C, D, E, F and G beta strands and the N-terminal region (amino acids corresponding to residues 1-7 of SEQ ID NO: 1) and the C-terminal region (amino acids corresponding to residues 93-94 of SEQ ID NO: 1).

"Amino acid sequence identity percentage (%)" herein is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the selected sequence after alignment of the sequences and introduction of gaps (if necessary, to achieve the maximum sequence identity percentage, and not considering any conservative substitutions as part of the sequence identity). Alignment for determining percentage of amino acid sequence identity can be achieved in various ways within the skill in the art, for example, using publicly available computer software such as BLAST ^SM , BLAST ^SM -2, ALIGN, ALIGN-2 or Megalign Software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

For the purpose of this article, the amino acid sequence identity % of a given amino acid sequence A relative to (with, or for) a given amino acid sequence B (or it can be expressed as a given amino acid sequence A relative to (with, or for) a given amino acid sequence B has or contains a certain amino acid sequence identity %) is calculated as follows: 100 multiplied by a fraction X/Y, where X is the sequence alignment program of A and B (such as BLAST ^SM , BLAST ^SM -2, ALIGN, ALIGN-2 or Megalign ) alignment that are scored as identical matches by the program, and where Y is the total number of amino acid residues in B. It will be understood that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A.

As used herein, the term "Adnectin binding site" refers to a site or portion of a protein (e.g., GPC3) that interacts or binds to a specific Adnectin. An Adnectin binding site can be formed by contiguous amino acids or non-contiguous amino acids juxtaposed by the tertiary folding of the protein. Adnectin binding sites formed by contiguous amino acids are generally retained when exposed to denaturing solvents, while Adnectin binding sites formed by tertiary folding are generally lost when treated with denaturing solvents.

The Adnectin binding sites of the anti-GPC3 Adnectins described herein can be determined by applying standard techniques commonly used for epitope mapping of antibodies, including, but not limited to, protease mapping and mutational analysis.

As used herein, an amino acid residue in a polypeptide is considered to "contribute to binding" to a target if (1) any non-hydrogen atom of the side chain or backbone of the residue is found to be within five angstroms of any atom of the bound target on the basis of the experimentally determined three-dimensional structure of the complex, and/or (2) mutation of the residue to its equivalent in wild- ^type10Fn3 (e.g., SEQ ID NO: 1), to alanine, or to a residue with a side chain of similar size or smaller than the residue in question, results in an increase in the measured equilibrium dissociation constant for the target (e.g., an increase _in kon).

In the context of FBS binding to GPC3, the terms "specifically binds," "specifically binds," "selectively binds," and "selectively binds," as used interchangeably herein, refer to a FBS that exhibits affinity for GPC3 but does not significantly bind (e.g., less than about 10% binding) to a different polypeptide, as measured by techniques available in the art, such as, but not limited to, Scatchard analysis and/or competitive binding assays (e.g., competition ELISA, BIACORE assays). The terms are also applicable where, for example, the binding domain of the FBS described herein is specific for GPC3 from one or more species (e.g., human, rodent, primate) but does not cross-react with other glypicans (e.g., glypican 1, glypican 2, glypican 5, glypican 6).

In the context of Adnectins that bind to GPC3, the term "preferentially binds" as used herein refers to situations where the binding of an Adnectin described herein to GPC3 is at least about 20% greater than its binding to a different polypeptide, as measured by techniques available in the art, such as, but not limited to, Scatchard analysis and/or competitive binding assays (e.g., competition ELISA, BIACORE assay).

In the context of Adnectins that bind to GPC3, the term " _KD " as used herein means the dissociation equilibrium constant of a particular Adnectin-protein (e.g., GPC3) interaction or the affinity of an Adnectin for a protein (e.g., GPC3), as measured using a surface plasmon resonance assay or a cell binding assay. As used herein, "desired _KD " refers to a _KD of an Adnectin that is sufficient for the intended purpose. For example, a desired _KD may refer to the _KD of an Adnectin required to elicit a functional effect in an in vitro assay, such as a cell-based luciferase assay.

In the context of an Adnectin bound to a protein, the term " _ka " as used herein refers to the association rate constant for the binding of the Adnectin into the Adnectin/protein complex.

In the context of an Adnectin bound to a protein, the term " _kd " as used herein refers to the dissociation rate constant for the dissociation of the Adnectin from the Adnectin/protein complex.

In the context of Adnectins, the term " _IC50 " as used herein refers to the concentration of Adnectin that inhibits a response in an in vitro or in vivo assay to a level that is 50% of the maximal inhibitory response (ie, halfway between the maximal inhibitory response and the untreated response).

As used herein, the term "glypican activity" or "glypican 3" activity refers to one or more growth regulating activities or morphogenic activities associated with GPC3 activation of cell signaling (e.g., activation of Wnt signaling). For example, GPC3 can regulate tumor cell growth by forming a complex with Wnt and/or Frizzled proteins. GPC3 can also activate signal transduction pathways and tumor cell growth by interacting with FGF. GPC3 activity can be determined using art-recognized methods, such as those described herein. The phrases "glypican 3 activity" or "antagonize glypican 3 activity" or "antagonize glypican 3" are used interchangeably and refer to the ability of the anti-GPC3 FBS and anti-GPC3 drug conjugates provided herein to neutralize or antagonize the activity of GPC3 in vivo or in vitro. As used herein, the term "inhibit" or "neutralize" with respect to anti-GPC3FBS activity refers to the ability to substantially antagonize, prevent, inhibit, slow, destroy, eliminate, stop, reduce or reverse, for example, the progression or severity of an inhibited entity, including but not limited to biological activities or properties, diseases or disorders (e.g., tumor cell growth). Inhibition or neutralization is preferably at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more.

As used herein, the term "connection" refers to the combination of two or more molecules. The connection can be covalent or non-covalent. The connection can be between a polypeptide and a chemical module or another polypeptide module. The connection can also be genetic (i.e., recombinant fusion). Such connection can be achieved using a variety of techniques recognized in the art, such as chemical conjugation and recombinant protein production.

The term "PK" is an acronym for "pharmacokinetics", which encompasses the properties of a compound, including, for example, absorption, distribution, metabolism and elimination by a subject. As used herein, a "PK modulator" or "PK module" refers to any protein, peptide or module that affects the pharmacokinetic properties of a bioactive molecule when fused to or administered with a bioactive molecule. Examples of PK modulators or PK modules include PEG, human serum albumin (HSA) binding agents (such as disclosed in U.S. Publication Nos. 2005/0287153 and 2007/0003549, PCT Publication Nos. WO 2009/083804 and WO 2009/133208), human serum albumin and variants thereof, transferrin and variants thereof, Fc or Fc fragments and variants thereof, and carbohydrates (e.g., sialic acid).

The serum or plasma "half-life" of a polypeptide may generally be defined as the time taken for the serum concentration of the polypeptide to decrease by 50% in vivo, e.g., due to degradation of the polypeptide by natural mechanisms and/or clearance or sequestration of the polypeptide. The half-life may be determined in any manner known per se, such as by pharmacokinetic analysis. Suitable techniques will be clear to those skilled in the art and may, for example, generally involve the following steps: administering a suitable dose of the polypeptide to a primate; collecting blood or other samples from the primate at regular intervals; determining the level of polypeptide concentration in the blood sample; and calculating, from (a plot of) the data thus obtained, the time until the level or concentration of the polypeptide decreases by 50% compared to the initial level at the time of administration. Methods for determining half-life can be found, for example, in Kenneth et al., Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists (1986); Peters et al., Pharmacokinete Analysis: A Practical Approach (1996); and Gibaldi, M. et al., Pharmacokinetics, Second Rev. Edition, Marcel Dekker (1982).

Serum half-life can be expressed using parameters such as t _1/2 -α, t _1/2 -β, and area under the curve (AUC). "Increase in half-life" refers to an increase in any one of these parameters, any two of these parameters, or all three of these parameters. In certain embodiments, an increase in half-life refers to an increase in t _1/2 -β and/or HL_λ_z, with or without an increase in t _1/2 -α and/or AUC or both.

The term "detectable" refers to the ability to detect a signal from a background signal. The term "detectable signal" is a signal derived from a non-invasive imaging technique (such as, but not limited to, positron emission tomography (PET)). The detectable signal can be detected and distinguished from other background signals that may be generated by the subject. In other words, there is a measurable and statistically significant difference between the detectable signal and the background (for example, a statistically significant difference is a difference sufficient to be distinguished in, such as a difference of about 0.1%, 1%, 3%, 5%, 10%, 15%, 20%, 25%, 30% or 40% or more between the detectable signal and the background). Standards and/or calibration curves can be used to determine the relative intensity of the detectable signal and/or background.

A "detectionally effective amount" of a composition comprising an imaging agent described herein is defined as an amount sufficient to produce an acceptable image using a device that can be used for clinical use. The detection effective amount of the imaging agent provided herein can be administered by more than one injection. The detection effective amount may vary according to factors such as the degree of susceptibility of the individual, the age, sex and weight of the individual, the specific reaction of the individual, etc. The detection effective amount of the imaging composition may also vary according to the instruments and methods used. The optimization of these factors is entirely within the technical level of the art. In certain embodiments, GPC3 imaging agents (e.g., those described herein) provide 2 or more (e.g., 3, 4, 5 or more) distinguishing factors (i.e., specific signals for background signals).

The terms "individual," "subject," and "patient," used interchangeably herein, refer to an animal, preferably a mammal (including non-primates and primates), such as a human.

"Cancer" refers to a broad variety of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth (dividing and growing) leads to the formation of malignant tumors, which invade adjacent tissues and may also metastasize to distant parts of the body via the lymphatic system or bloodstream.

"Treatment" or "therapy" of a subject refers to any type of intervention or procedure performed on a subject or in which an active agent is administered to the subject in order to reverse, alleviate, ameliorate, inhibit, slow or prevent the onset, progression, development, severity or recurrence of symptoms, complications, conditions or biochemical markers associated with a disease.

In the context of anti-GPC3 Adnectins, "administration" or "application" as used herein refers to the introduction of a GPC3 Adnectin or a GPC3 Adnectin-based probe or labeled probe (also referred to as an "imaging agent") described herein into a subject. Any suitable route of administration may be used, such as intravenous, oral, topical, subcutaneous, intraperitoneal, intraarterial, inhalation, vaginal, rectal, nasal, introduction into the cerebrospinal fluid, or instillation into a body compartment.

As used herein, the terms "co-administered," "co-administered," and the like are intended to include administration of the selected agents to a single patient, and are intended to include regimens in which the agents are administered by the same or different routes of administration, or at the same or different times.

The term "therapeutically effective amount" refers to at least the minimum dose of an agent necessary to confer a therapeutic benefit on a subject, but less than a toxic dose.

As used herein, an "effective amount" refers to at least an amount effective, at dosages and for periods of time, to achieve the desired result.

As used herein, "sufficient amount" refers to an amount sufficient to achieve a desired result.

The term "sample" may refer to a tissue sample, a cell sample, a fluid sample, etc. A sample may be taken from a subject. Tissue samples may include hair (including hair roots), oral swabs, blood, saliva, semen, muscle, or from any internal organ. Fluids may be, but are not limited to, urine, blood, ascites, pleural fluid, spinal fluid, etc. Body tissues may include, but are not limited to, skin, muscle, endometrium, uterus, and cervical tissue.

Overview

Provided herein are novel fibronectin-based scaffold polypeptides that bind human Glypican 3. Such polypeptides can be coupled to other therapeutic and diagnostic agents and can be used, for example, to target therapeutic and diagnostic agents to cells and tissues expressing Glypican 3 (e.g., cancer cells that overexpress Glypican 3).

I. Anti-GPC3 Fibronectin-based Scaffolds

As used herein, a "fibronectin-based scaffold" or "FBS" protein or module refers to a protein or module that is based on fibronectin type III ("Fn3") repeats and can be modified to specifically bind to a given target (e.g., a target protein). Fn3 is a small (about 10 kDa) domain with an immunoglobulin (Ig) fold (i.e., an Ig-like β sandwich structure consisting of seven β strands and six loops). Fibronectin has 18 Fn3 repeats, and although the sequence homology between the repeats is low, they all share a high degree of similarity in the tertiary structure. Fn3 domains also appear in a variety of proteins other than fibronectin, such as adhesion molecules, cell surface molecules (e.g., cytokine receptors), and carbohydrate binding domains. For review, see Bork et al., Proc. Natl. Acad. Sci. USA, 89(19):8990-8994 (1992); Bork et al., J. Mol. Biol., 242(4):309-320 (1994); Campbell et al., Structure, 2(5):333-337 (1994); Harpez et al., J. Mol. Biol., 238(4):528-539 (1994)). The term "FBS" protein or module is intended to include scaffolds based on Fn3 domains from these other proteins (i.e., non-fibronectin molecules).

The Fn3 domain is small, monomeric, soluble, and stable. It lacks disulfide bonds and is therefore stable under reducing conditions. In order from N-terminus to C-terminus, the Fn3 domain comprises β or β-like chain A; loop AB; β or β-like chain B; loop BC; β or β-like chain C; loop CD; β or β-like chain D; loop DE; β or β-like chain E; loop EF; β or β-like chain F; loop FG; and β or β-like chain G. Seven antiparallel β chains are arranged into two β sheets, which form a stable core while producing two "faces" consisting of loops connecting β or β-like chains. Loops AB, CD, and EF are located on one face ("South Pole") and loops BC, DE, and FG are located on the opposite face ("North Pole").

The loops in the Fn3 molecule are structurally similar to the complementary determining regions (CDRs) of antibodies and may be involved in the binding of the Fn3 molecule to a target, such as a target protein, when altered. Other regions of the Fn3 molecule, such as β or β-like strands and N-terminal or C-terminal regions, may be involved in the binding to the target when altered. Any or all of the loops AB, BC, CD, DE, EF and FG may participate in the binding to the target. Any β or β-like strand may be involved in the binding to the target. The Fn3 domain may also bind to the target through one or more loops and one or more β or β-like strands. The binding may also require the N-terminal or C-terminal region.

The anti-GPC3 FBS may be based on the tenth domain of fibronectin type III, i.e., the tenth module of human Fn3 ( ^10Fn3 ) in which one or more solvent accessible loops are randomized or mutated. The amino acid sequence of the wild-type human ^10Fn3 portion is as follows:

VSDVPRDLEVVAA TPTS LLISWDAPAVTVRYYRITY GETGGNSPVQE FTVPGSKSTATISGL KPGVD YTITVYAVTGRGDSPASSKPISINYRT(SEQ ID NO:1)

The AB, CD, and EF loops are underlined; the BC, FG, and DE loops are highlighted in bold; the beta strands are located between or near the various loop regions; and the N-terminal region is in italics). The last two amino acid residues of SEQ ID NO: 1 are part of the C-terminal region. The ^core10Fn3 domain begins with amino acid 9 ("E") and ends with amino acid 94 ("T"), and corresponds to an 86 amino acid polypeptide. The core wild-type ^human10Fn3 domain is set forth in SEQ ID NO: 2. Both the variant and wild- ^type10Fn3 proteins are characterized by the same structure, i.e., seven beta strand domain sequences designated A to G and six loop regions (AB loop, BC loop, CD loop, DE loop, EF loop, and FG loop) connecting the seven beta strand domain sequences. The beta strands located closest to the N-terminus and C-terminus can adopt a beta-like solution conformation. In SEQ ID NO: 1, the AB loop corresponds to residues 14-17, the BC loop corresponds to residues 23-31, the CD loop corresponds to residues 37-47, the DE loop corresponds to residues 51-56, the EF loop corresponds to residues 63-67, and the FG loop corresponds to residues 76-87.

Thus, in certain embodiments, ^an anti-GPC3 FBS moiety (e.g., an anti-GPC3 Adnectin) comprises ^a10Fn3 domain, which is generally defined by the following degenerate sequence:

VSDVPRD LEVVAA (X) _u LLISW (X) _v YRITY (X) _w FTV (X) _x ATISGL (X) _y YTITV YA (X) _z ISINY RT (SEQ ID NO:3), or is defined by a sequence lacking 1, 2, 3, 4, 5, 6 or 7 N-terminal amino acids, respectively.

In SEQ ID NO:3, the AB loop is represented by (X) _u , the BC loop is represented by (X) _v , the CD loop is represented by (X) _w , the DE loop is represented by (X) _x , the EF loop is represented by (X) _y , and the FG loop is represented by X _z . X represents any amino acid, and the subscript after X represents an integer of the number of amino acids. Specifically, u, v, w, x, y and z can each independently be any one selected from 2-20, 2-15, 2-10, 2-8, 5-20, 5-15, 5-10, 5-8, 6-20, 6-15, 6-10, 6-8, 2-7, 5-7, or 6-7 amino acids. The beta strand sequence (underlined in SEQ ID NO: 3) can have any of from 0 to 10, from 0 to 8, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1 substitutions, deletions, or additions across all 7 scaffold regions relative to the corresponding amino acids shown in SEQ ID NO: 3. In some embodiments, the beta strand sequence can have any of from 0 to 10, from 0 to 8, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1 substitutions (e.g., conservative substitutions) across all 7 scaffold regions relative to the corresponding amino acids shown in SEQ ID NO: 3.

It should be understood that in order to achieve ^a10Fn3 binding domain with strong affinity for the desired target, not every residue in the loop region needs to be modified or changed. In addition, insertions and deletions can also be formed in the loop region while still producing a high-affinity anti- ^GPC310Fn3 binding domain. By "alteration" is meant one or more amino acid sequence changes relative to the template sequence (i.e., the corresponding wild-type human fibronectin domain) and includes amino acid additions, deletions, substitutions, and combinations thereof.

In some embodiments, the anti-GPC3 FBS moiety comprises ^a10Fn3 domain, wherein the non-loop region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to the non-loop region of SEQ ID NO: 1, and wherein at least one loop selected from AB, BC, CD, DE, EF, and FG is altered.

In some embodiments, one or more loops selected from AB, BC, CD, DE, EF and FG can be extended or shortened in length relative to the corresponding loops in wild-type ^human10Fn3 . In any given polypeptide, one or more loops can be extended in length, one or more loops can be shortened in length, or a combination of the two. In some embodiments, the length of a given loop can be extended by 2-25, 2-20, 2-15, 2-10, 2-5, 5-25, 5-20, 5-15, 5-10, 10-25, 10-20, or 10-15 amino acids. In some embodiments, the length of a given loop can be shortened by 1-15, 1-11, 1-10, 1-5, 1-3, 1-2, 2-10, or 2-5 amino acids. Specifically, the length of the FG loop ^of10Fn3 is 13 residues, while the length range of the corresponding loop in the antibody heavy chain is 4-28 residues. Thus, in some embodiments, the length as well as the sequence of the FG loop ^of10Fn3 can be altered to achieve the greatest possible flexibility and target affinity in a polypeptide whose target binding is dependent on the FG loop.

In certain embodiments, the anti-GPC3 FBS module comprises a fibronectin type III tenth ( ^10Fn3 ) domain, wherein ^the10Fn3 domain comprises loop AB, loop BC, loop CD, loop DE, loop EF, and loop FG, and wherein at least one loop selected from loop ^BC , DE, and FG has an altered amino acid sequence relative to the corresponding loop sequence of a ^human10Fn3 domain. In some embodiments, the anti-GPC3 Adnectin described herein comprises ^a10Fn3 domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to a non-loop region of SEQ ID NO: 1 or 2, and wherein at least one loop selected from BC, DE, and FG is altered. In certain embodiments, the BC and FG loops are altered, in certain embodiments, the BC and DE loops are altered, in certain embodiments, the DE and FG loops are altered, and in certain embodiments, the BC, DE, and FG loops are altered, i.e., ^the10Fn3 domain comprises non-naturally occurring loops. In certain embodiments, the AB, CD and/or EF loops are altered.In some embodiments, one or more prescribed scaffold alterations are combined with one or more loop alterations.

In certain embodiments, the non- ^ligand binding sequences of the anti-GPC310Fn3 may be altered provided that ^{the10Fn3 retains ligand binding function and/or structural stability. In some embodiments, the non-loop regions of the10Fn3 domain may be modified by one or more conservative substitutions. Up to 5%, 10 %} , 20%, or even 30% or more of the amino acids in ^the10Fn3 domain may be altered by conservative substitutions without substantially altering the ligand affinity of ^the10Fn3 . In certain embodiments, the non-loop regions, such as the beta strands, may comprise any one of 0-15, 0-10, 0-8, 0-6, 0-5, 0-4, 0-3, 1-15, 1-10, 1-8, 1-6, 1-5, 1-4, 1-3, 2-15, 2-10, 2-8, 2-6, 2-5, 2-4, 5-15, or 5-10 conservative amino acid substitutions. In exemplary embodiments, the scaffold modification may reduce the binding affinity of the ^10Fn3 binding agent to the ligand by less than 100-fold, 50-fold, 25-fold, 10-fold, 5-fold, or 2-fold. Such changes may alter the in vivo immunogenicity of ^10Fn3 , and in the case of reduced immunogenicity, such changes may be desirable. As used herein, a "conservative substitution" is a residue that is physically or functionally similar to a corresponding reference residue. That is, a conservative substitution has similar size, properties, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc., to its reference residue. Exemplary conservative substitutions include those that meet the criteria defined in Dayhoff et al., Atlas of Protein Sequence and Structure, 5:345-352 (1978 and Supp.) for acceptable point mutations. Examples of conservative substitutions include substitutions within the following groups: (a) valine, glycine; (b) glycine, alanine; (c) valine, isoleucine, leucine; (d) aspartic acid, glutamic acid; (e) asparagine, glutamine; (f) serine, threonine; (g) lysine, arginine, methionine; and (h) phenylalanine, tyrosine.

In some embodiments, one or more of Asp 7, Glu 9, and Asp 23 are replaced by another amino acid, such as, for example, a non-negatively charged amino acid residue (e.g., Asn, Lys, etc.). Various additional changes that are beneficial or neutral in the ¹⁰ Fn3 scaffold have been disclosed. See, for example, Batori et al., Protein Eng., 15(12): 1015-1020 (December 2002); Koide et al., Biochemistry, 40(34): 10326-10333 (Aug. 28, 2001).

In other embodiments, the hydrophobic core amino acid residues (the residues in bold in SEQ ID NO:3 above) are fixed, and any substitutions, conservative substitutions, deletions or additions occur at residues other than the hydrophobic core amino acid residues in ^the10Fn3 scaffold. Thus, in some embodiments, the hydrophobic core residues of the polypeptides provided herein are unmodified relative to the wild-type ^human10Fn3 domain (e.g., SEQ ID NO: 1).

^The10Fn3 molecule may comprise a flexible linker between the 10th and 11th repeats of the Fn3 domain, ie, EIDKPSQ (SEQ ID NO: 369). A wild- ^type10Fn3 polypeptide having EIDKPSQ (SEQ ID NO: 369) at its C-terminus is represented by the following sequence:

VSDVPRDLEVVAA TPTS LLISWDAPAVTVRYYRITY GETGGNSPVQE FTVPGSKSTATISGL KPGVD YTITVYAVTGRGDSPASSKPISINYRT EIDKPSQ(SEQ ID NO:4)

In some embodiments, one or more residues of the integrin binding motif "arginine-glycine-aspartic acid" (RGD) (amino acids 78-80 of SEQ ID NO: 1) can be replaced to disrupt integrin binding. In some embodiments, the FG loop of the polypeptide provided herein does not contain an RGD integrin binding site. In one embodiment, the RGD sequence is replaced with a polar amino acid-neutral amino acid-acidic amino acid sequence (in the direction of the N-terminus to the C-terminus). In certain embodiments, the RGD sequence is replaced with SGE or RGE.

A. Exemplary Anti-GPC3 Adnectins

In some embodiments, the BC loop of the anti-GPC3 FBS (e.g., an Adnectin that specifically binds to human GPC3) comprises the amino acid sequence set forth in SEQ ID NO:6, 19, 32, 45, 58, 71, 84, or 99, wherein optionally the BC loop comprises 1, 2, or 3 amino acid substitutions, deletions, or insertions relative to the BC loop of SEQ ID NO:6, 19, 32, 45, 58, 71, 84, or 99.

In some embodiments, the DE loop of the anti-GPC3 FBS comprises the amino acid sequence set forth in SEQ ID NO:7, 20, 33, 46, 59, 72, 85, or 100, wherein optionally, the DE loop comprises 1, 2, or 3 amino acid substitutions, deletions, or insertions relative to the DE loop of SEQ ID NO:7, 20, 33, 46, 59, 72, 85, or 100.

In some embodiments, the FG loop of the anti-GPC3 FBS comprises the amino acid sequence set forth in SEQ ID NO:8, 21, 34, 47, 60, 73, 86, 101, 129, 156, 183, 210, 237, 264, 291, or 318, wherein optionally, the FG loop comprises 1, 2, or 3 amino acid substitutions, deletions, or insertions relative to the FG loop of SEQ ID NO:8, 21, 34, 47, 60, 73, 86, 101, 129, 156, 183, 210, 237, 264, 291, or 318.

In some embodiments, the BC loop of the anti-GPC3 Adnectin (e.g., an Adnectin that specifically binds to human GPC3) comprises the amino acid sequence set forth in SEQ ID NO: 6, 19, 32, 45, 58, 71, 84, or 99, wherein optionally, the BC loop comprises 1, 2, or 3 amino acid substitutions, deletions, or insertions relative to the BC loop of SEQ ID NO: 6, 19, 32, 45, 58, 71, 84, or 99; the DE loop of the anti-GPC3 Adnectin comprises the amino acid sequence set forth in SEQ ID NO: 7, 20, 33, 46, 59, 72, 85, or 100, wherein optionally, the DE loop comprises 1, 2, or 3 amino acid substitutions, deletions, or insertions relative to the DE loop of SEQ ID NO: 7, 20, 33, 46, 59, 72, 85, or 100; and the FG loop of the anti-GPC3 FBS comprises the amino acid sequence set forth in SEQ ID NO: NO:8, 21, 34, 47, 60, 73, 86, 101, 129, 156, 183, 210, 237, 264, 291 or 318, wherein optionally, the FG loop comprises 1, 2 or 3 amino acid substitutions, deletions or insertions relative to the FG loop of SEQ ID NO:8, 21, 34, 47, 60, 73, 86, 101, 129, 156, 183, 210, 237, 264, 291 or 318.

In some embodiments, the anti-GPC3 FBS comprises: a BC loop comprising the amino acid sequence set forth in SEQ ID NO:6, 19, 32, 45, 58, 71, 84, or 99; a DE loop comprising the amino acid sequence set forth in SEQ ID NO:7, 20, 33, 46, 59, 72, 85, or 100; and a FG loop comprising the amino acid sequence set forth in SEQ ID NO:8, 21, 34, 47, 60, 73, 86, 101, 129, 156, 183, 210, 237, 264, 291, or 318.

In some embodiments, an anti-GPC3 FBS comprises the BC, DE, and FG loops set forth in SEQ ID NOs: 6, 19, 32, 45, 58, 71, 84, or 99; 7, 20, 33, 46, 59, 72, 85, or 100; and 8, 21, 34, 47, 60, 73, 86, 101, 129, 156, 183, 210, 237, 264, 291, or 318, respectively, and has amino acid substitutions in the BC, DE, and FG loops that allow the FBS to maintain binding to GPC3.

In some embodiments, the anti-GPC3 FBS comprises the amino acid sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops represented by (X) _v , (X) _x , and (X) _z, respectively, have amino acid sequences that are at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the BC, DE, or FG loop sequences set forth in SEQ ID NOs:6, 7, and 8, respectively.

In some embodiments, the anti-GPC3 Adnectin comprises the amino acid sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops comprise the amino acid sequences set forth in SEQ ID NOs:6, 7, and 8, respectively, wherein the BC loop has 0, 1, 2, 3, 4, 5, or 6 amino acid substitutions, such as conservative amino acid substitutions, and the DE loop has 0, 1, 2, or 3 amino acid substitutions, such as conservative amino acid substitutions, and the FG loop has 0, 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions, such as conservative amino acid substitutions.

In certain embodiments, the anti-GPC3 Adnectin (e.g., an anti-FBS moiety comprising ^human10Fn3 ) comprises the sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops represented by (X) _v , (X) _x , and (X) _z, respectively, include BC, DE, and FG loops having the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively.

In certain embodiments, the anti-GPC3 Adnectin comprises the sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops represented by (X) _v , (X) _x , and (X) _z, respectively, have an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the BC, DE, or FG loop sequences set forth in SEQ ID NOs:19, 20, and 21, respectively.

In some embodiments, the anti-GPC3 Adnectin comprises the amino acid sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops comprise the amino acid sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively, wherein the BC loop has 0, 1, 2, 3, 4, 5, or 6 amino acid substitutions, such as conservative amino acid substitutions, and the DE loop has 0, 1, 2, or 3 amino acid substitutions, such as conservative amino acid substitutions, and the FG loop has 0, 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions, such as conservative amino acid substitutions.

In certain embodiments, the anti-GPC3 Adnectin comprises the sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops represented by (X) _v , (X) _x , and (X) _{z ,} respectively, include BC, DE, and FG loops having the amino acid sequences of SEQ ID NOs: 19, 20, and 21 , respectively.

In certain embodiments, the anti-GPC3 Adnectin comprises the sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops represented by (X) _v , (X) _x , and (X) _z, respectively, have an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the BC, DE, or FG loop sequences set forth in SEQ ID NOs:32, 33, and 34, respectively.

In some embodiments, the anti-GPC3 Adnectin comprises the amino acid sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops comprise the amino acid sequences set forth in SEQ ID NOs:32, 33, and 34, respectively, wherein the BC loop has 0, 1, 2, 3, 4, 5, or 6 amino acid substitutions, such as conservative amino acid substitutions, and the DE loop has 0, 1, 2, or 3 amino acid substitutions, such as conservative amino acid substitutions, and the FG loop has 0, 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions, such as conservative amino acid substitutions.

In certain embodiments, the anti-GPC3 Adnectin comprises the sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops represented by (X) _v , (X) _x , and (X) _{z ,} respectively, comprise BC, DE, and FG loops having the amino acid sequences of SEQ ID NOs: 32, 33, and 34, respectively.

In certain embodiments, the anti-GPC3 Adnectin comprises the sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops represented by (X) _v , (X) _x , and (X) _z, respectively, have an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the BC, DE, or FG loop sequences set forth in SEQ ID NOs:45, 46, and 47, respectively.

In some embodiments, the anti-GPC3 Adnectin comprises the amino acid sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops comprise the amino acid sequences set forth in SEQ ID NOs:45, 46, and 47, respectively, wherein the BC loop has 0, 1, 2, 3, 4, 5, or 6 amino acid substitutions, such as conservative amino acid substitutions, and the DE loop has 0, 1, 2, or 3 amino acid substitutions, such as conservative amino acid substitutions, and the FG loop has 0, 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions, such as conservative amino acid substitutions.

In certain embodiments, the anti-GPC3 Adnectin comprises the sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops represented by (X) _v , (X) _x , and (X) _{z ,} respectively, include BC, DE, and FG loops having the amino acid sequences of SEQ ID NOs: 45, 46, and 47, respectively.

In certain embodiments, the anti-GPC3 Adnectin comprises the sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops represented by (X) _v , (X) _x , and (X) _z, respectively, have an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the BC, DE, or FG loop sequences set forth in SEQ ID NOs:58, 59, and 60, respectively.

In some embodiments, the anti-GPC3 Adnectin comprises the amino acid sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops comprise the amino acid sequences set forth in SEQ ID NOs:58, 59, and 60, respectively, wherein the BC loop has 0, 1, 2, 3, 4, 5, or 6 amino acid substitutions, such as conservative amino acid substitutions, and the DE loop has 0, 1, 2, or 3 amino acid substitutions, such as conservative amino acid substitutions, and the FG loop has 0, 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions, such as conservative amino acid substitutions.

In certain embodiments, the anti-GPC3 Adnectin comprises the sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops represented by (X) _v , (X) _x , and (X) _{z ,} respectively, comprise BC, DE, and FG loops having the amino acid sequences of SEQ ID NOs: 58, 59, and 60, respectively.

In certain embodiments, the anti-GPC3 Adnectin comprises the sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops represented by (X) _v , (X) _x , and (X) _z, respectively, have an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the BC, DE, or FG loop sequences set forth in SEQ ID NOs:71, 72, and 73, respectively.

In some embodiments, the anti-GPC3 Adnectin comprises the amino acid sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops comprise the amino acid sequences set forth in SEQ ID NOs:71, 72, and 73, respectively, wherein the BC loop has 0, 1, 2, 3, 4, 5, or 6 amino acid substitutions, such as conservative amino acid substitutions, and the DE loop has 0, 1, 2, or 3 amino acid substitutions, such as conservative amino acid substitutions, and the FG loop has 0, 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions, such as conservative amino acid substitutions.

In certain embodiments, the anti-GPC3 Adnectin comprises the sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops represented by (X) _v , (X) _x , and (X) _{z ,} respectively, include BC, DE, and FG loops having the amino acid sequences of SEQ ID NOs: 71 , 72, and 73, respectively.

In certain embodiments, the anti-GPC3 Adnectin comprises the sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops represented by (X) _v , (X) _x , and (X) _z, respectively, have an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the BC, DE, or FG loop sequences set forth in SEQ ID NOs:84, 85, and 86, respectively.

In some embodiments, the anti-GPC3 Adnectin comprises the amino acid sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops comprise the amino acid sequences set forth in SEQ ID NOs:84, 85, and 86, respectively, wherein the BC loop has 0, 1, 2, 3, 4, 5, or 6 amino acid substitutions, such as conservative amino acid substitutions, and the DE loop has 0, 1, 2, or 3 amino acid substitutions, such as conservative amino acid substitutions, and the FG loop has 0, 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions, such as conservative amino acid substitutions.

In certain embodiments, the anti-GPC3 Adnectin comprises the sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops represented by (X) _v , (X) _x , and (X) _{z ,} respectively, comprise BC, DE, and FG loops having the amino acid sequences of SEQ ID NOs: 84, 85, and 86, respectively.

In certain embodiments, the anti-GPC3 Adnectin comprises the sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops represented by (X) _v , (X) _x , and (X) _z, respectively, have an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the BC, DE, or FG loop sequences set forth in SEQ ID NOs:99, 100, and 101, respectively.

In some embodiments, the anti-GPC3 Adnectin comprises the amino acid sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops comprise the amino acid sequences set forth in SEQ ID NOs:99, 100, and 101, respectively, wherein the BC loop has 0, 1, 2, 3, 4, 5, or 6 amino acid substitutions, such as conservative amino acid substitutions, and the DE loop has 0, 1, 2, or 3 amino acid substitutions, such as conservative amino acid substitutions, and the FG loop has 0, 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions, such as conservative amino acid substitutions.

In certain embodiments, the anti-GPC3 Adnectin comprises the sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops represented by (X) _v , (X) _x , and (X) _{z ,} respectively, include BC, DE, and FG loops having the amino acid sequences of SEQ ID NOs: 99, 100, and 101 , respectively.

In some embodiments, the anti-GPC3 Adnectin comprises the amino acid sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops comprise the amino acid sequences set forth in SEQ ID NOs:99, 100, and 129, respectively, wherein the BC loop has 0, 1, 2, 3, 4, 5, or 6 amino acid substitutions, such as conservative amino acid substitutions, and the DE loop has 0, 1, 2, or 3 amino acid substitutions, such as conservative amino acid substitutions.

In certain embodiments, the anti-GPC3 Adnectin comprises the sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops represented by (X) _v , (X) _x , and (X) _{z ,} respectively, include BC, DE, and FG loops having amino acid sequences of SEQ ID NOs: 99, 100, and 129, respectively.

In some embodiments, the anti-GPC3 Adnectin comprises the amino acid sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops comprise the amino acid sequences set forth in SEQ ID NOs:99, 100, and 156, respectively, wherein the BC loop has 0, 1, 2, 3, 4, 5, or 6 amino acid substitutions, such as conservative amino acid substitutions, and the DE loop has 0, 1, 2, or 3 amino acid substitutions, such as conservative amino acid substitutions.

In certain embodiments, the anti-GPC3 Adnectin comprises the sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops represented by (X) _v , (X) _x , and (X) _{z ,} respectively, include BC, DE, and FG loops having amino acid sequences of SEQ ID NOs: 99, 100, and 156, respectively.

In some embodiments, the anti-GPC3 Adnectin comprises the amino acid sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops comprise the amino acid sequences set forth in SEQ ID NOs:99, 100, and 183, respectively, wherein the BC loop has 0, 1, 2, 3, 4, 5, or 6 amino acid substitutions, such as conservative amino acid substitutions, and the DE loop has 0, 1, 2, or 3 amino acid substitutions, such as conservative amino acid substitutions.

In certain embodiments, the anti-GPC3 Adnectin comprises the sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops represented by (X) _v , (X) _x , and (X) _{z ,} respectively, include BC, DE, and FG loops having amino acid sequences of SEQ ID NOs: 99, 100, and 183, respectively.

In some embodiments, the anti-GPC3 Adnectin comprises the amino acid sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops comprise the amino acid sequences set forth in SEQ ID NOs:99, 100, and 210, respectively, wherein the BC loop has 0, 1, 2, 3, 4, 5, or 6 amino acid substitutions, such as conservative amino acid substitutions, and the DE loop has 0, 1, 2, or 3 amino acid substitutions, such as conservative amino acid substitutions.

In certain embodiments, the anti-GPC3 Adnectin comprises the sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops represented by (X) _v , (X) _x , and (X) _{z ,} respectively, include BC, DE, and FG loops having amino acid sequences of SEQ ID NOs: 99, 100, and 210, respectively.

In some embodiments, the anti-GPC3 Adnectin comprises the amino acid sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops comprise the amino acid sequences set forth in SEQ ID NOs:99, 100, and 237, respectively, wherein the BC loop has 0, 1, 2, 3, 4, 5, or 6 amino acid substitutions, such as conservative amino acid substitutions, and the DE loop has 0, 1, 2, or 3 amino acid substitutions, such as conservative amino acid substitutions.

In certain embodiments, the anti-GPC3 Adnectin comprises the sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops represented by (X) _v , (X) _x , and (X) _{z ,} respectively, include BC, DE, and FG loops having amino acid sequences of SEQ ID NOs: 99, 100, and 237, respectively.

In some embodiments, the anti-GPC3 Adnectin comprises the amino acid sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops comprise the amino acid sequences set forth in SEQ ID NOs:99, 100, and 264, respectively, wherein the BC loop has 0, 1, 2, 3, 4, 5, or 6 amino acid substitutions, such as conservative amino acid substitutions, and the DE loop has 0, 1, 2, or 3 amino acid substitutions, such as conservative amino acid substitutions.

In certain embodiments, the anti-GPC3 Adnectin comprises the sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops represented by (X) _v , (X) _x , and (X) _{z ,} respectively, comprise BC, DE, and FG loops having amino acid sequences of SEQ ID NOs: 99, 100, and 264, respectively.

In some embodiments, the anti-GPC3 Adnectin comprises the amino acid sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops comprise the amino acid sequences set forth in SEQ ID NOs:99, 100, and 291, respectively, wherein the BC loop has 0, 1, 2, 3, 4, 5, or 6 amino acid substitutions, such as conservative amino acid substitutions, and the DE loop has 0, 1, 2, or 3 amino acid substitutions, such as conservative amino acid substitutions.

In certain embodiments, the anti-GPC3 Adnectin comprises the sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops represented by (X) _v , (X) _x , and (X) _{z ,} respectively, include BC, DE, and FG loops having amino acid sequences of SEQ ID NOs: 99, 100, and 291 , respectively.

In some embodiments, the anti-GPC3 Adnectin comprises the amino acid sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops comprise the amino acid sequences set forth in SEQ ID NOs:99, 100, and 318, respectively, wherein the BC loop has 0, 1, 2, 3, 4, 5, or 6 amino acid substitutions, such as conservative amino acid substitutions, and the DE loop has 0, 1, 2, or 3 amino acid substitutions, such as conservative amino acid substitutions.

In certain embodiments, the anti-GPC3 Adnectin comprises the sequence set forth in SEQ ID NO:3, wherein the BC, DE, and FG loops represented by (X) _v , (X) _x , and (X) _{z ,} respectively, comprise BC, DE, and FG loops having amino acid sequences of SEQ ID NOs: 99, 100, and 318, respectively.

The scaffold region of such an anti-GPC3 Adnectin may comprise any one of from 0 to 20, from 0 to 15, from 0 to 10, from 0 to 8, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1 substitutions, conservative substitutions, deletions or additions relative to the scaffold amino acid residues of SEQ ID NO: 3. Such scaffold modifications may be made as long as the anti-GPC3 Adnectin is able to bind to GPC3 with a desired _KD .

In certain embodiments, the anti-GPC3 Adnectin comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to an anti-GPC3 Adnectin disclosed herein and has, for example, any one of SEQ ID NOs: 5, 18, 31, 44, 57, 70, 83, and 98.

In certain embodiments, the anti-GPC3 Adnectin comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 5, 9-18, 22-31, 35-44, 48-57, 61-70, 74-83, 87-98, 102-128, 130-155, 157-182, 184-209, 211-236, 238-263, 265-290, 292-317, and 319-343.

In certain embodiments, the anti-GPC3 Adnectins described herein comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the non-BC, DE and FG loop regions of SEQ ID NO:3, 5, 18, 31, 44, 57, 70, 83 or 98.

In certain embodiments, the anti-GPC3 Adnectin comprises the BC, DE, and FG loops as set forth in SEQ ID NOs: 6, 7, and 8, respectively; and an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the non-BC, DE, and FG loop regions of SEQ ID NOs: 3, 5, 18, 31, 44, 57, 70, 83, or 98.

In certain embodiments, the anti-GPC3 Adnectin comprises the BC, DE, and FG loops as set forth in SEQ ID NOs: 19, 20, and 21, respectively; and an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the non-BC, DE, and FG loop regions of SEQ ID NOs: 3, 5, 18, 31, 44, 57, 70, 83, or 98.

In certain embodiments, the anti-GPC3 Adnectin comprises the BC, DE, and FG loops as set forth in SEQ ID NOs: 32, 33, and 34, respectively; and an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the non-BC, DE, and FG loop regions of SEQ ID NOs: 3, 5, 18, 31, 44, 57, 70, 83, or 98.

In certain embodiments, the anti-GPC3 Adnectin comprises the BC, DE, and FG loops as set forth in SEQ ID NOs: 45, 46, and 47, respectively; and an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the non-BC, DE, and FG loop regions of SEQ ID NOs: 3, 5, 18, 31, 44, 57, 70, 83, or 98.

In certain embodiments, the anti-GPC3 Adnectin comprises the BC, DE, and FG loops as set forth in SEQ ID NOs: 58, 59, and 60, respectively; and an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the non-BC, DE, and FG loop regions of SEQ ID NOs: 3, 5, 18, 31, 44, 57, 70, 83, or 98.

In certain embodiments, the anti-GPC3 Adnectin comprises the BC, DE, and FG loops as set forth in SEQ ID NOs:71, 72, and 73, respectively; and an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the non-BC, DE, and FG loop regions of SEQ ID NOs:3, 5, 18, 31, 44, 57, 70, 83, or 98.

In certain embodiments, the anti-GPC3 Adnectin comprises the BC, DE, and FG loops as set forth in SEQ ID NOs: 84, 85, and 86, respectively; and an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the non-BC, DE, and FG loop regions of SEQ ID NOs: 3, 5, 18, 31, 44, 57, 70, 83, or 98.

In certain embodiments, the anti-GPC3 Adnectin comprises the BC, DE, and FG loops as set forth in SEQ ID NOs: 99, 100, and 101, respectively; and an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the non-BC, DE, and FG loop regions of SEQ ID NOs: 3, 5, 18, 31, 44, 57, 70, 83, or 98.

In certain embodiments, the anti-GPC3 Adnectin comprises the BC, DE, and FG loops as set forth in SEQ ID NOs: 99, 100, and 129, respectively; and an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the non-BC, DE, and FG loop regions of SEQ ID NOs: 3, 5, 18, 31, 44, 57, 70, 83, or 98.

In certain embodiments, the anti-GPC3 Adnectin comprises the BC, DE, and FG loops as set forth in SEQ ID NOs: 99, 100, and 129, respectively; and an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the non-BC, DE, and FG loop regions of SEQ ID NOs: 3, 5, 18, 31, 44, 57, 70, 83, or 98.

In certain embodiments, the anti-GPC3 Adnectin comprises the BC, DE, and FG loops as set forth in SEQ ID NOs: 99, 100, and 156, respectively; and an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the non-BC, DE, and FG loop regions of SEQ ID NOs: 3, 5, 18, 31, 44, 57, 70, 83, or 98.

In certain embodiments, the anti-GPC3 Adnectin comprises the BC, DE, and FG loops as set forth in SEQ ID NOs: 99, 100, and 183, respectively; and an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the non-BC, DE, and FG loop regions of SEQ ID NOs: 3, 5, 18, 31, 44, 57, 70, 83, or 98.

In certain embodiments, the anti-GPC3 Adnectin comprises the BC, DE, and FG loops as set forth in SEQ ID NOs: 99, 100, and 210, respectively; and an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the non-BC, DE, and FG loop regions of SEQ ID NOs: 3, 5, 18, 31, 44, 57, 70, 83, or 98.

In certain embodiments, the anti-GPC3 Adnectin comprises the BC, DE, and FG loops as set forth in SEQ ID NOs: 99, 100, and 237, respectively; and an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the non-BC, DE, and FG loop regions of SEQ ID NOs: 3, 5, 18, 31, 44, 57, 70, 83, or 98.

In certain embodiments, the anti-GPC3 Adnectin comprises the BC, DE, and FG loops as set forth in SEQ ID NOs: 99, 100, and 264, respectively; and an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the non-BC, DE, and FG loop regions of SEQ ID NOs: 3, 5, 18, 31, 44, 57, 70, 83, or 98.

In certain embodiments, the anti-GPC3 Adnectin comprises the BC, DE, and FG loops as set forth in SEQ ID NOs: 99, 100, and 291, respectively; and an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the non-BC, DE, and FG loop regions of SEQ ID NOs: 3, 5, 18, 31, 44, 57, 70, 83, or 98.

In certain embodiments, the anti-GPC3 Adnectin comprises the BC, DE, and FG loops as set forth in SEQ ID NOs: 99, 100, and 318, respectively; and an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the non-BC, DE, and FG loop regions of SEQ ID NOs: 3, 5, 18, 31, 44, 57, 70, 83, or 98.

In some embodiments, the anti-GPC3 Adnectin comprises an amino acid sequence selected from the group consisting of 5, 18, 31, 44, 57, 70, 83, 98, 128, 155, 182, 209, 209, 236, 263, 290, and 317.

In some embodiments, the anti-GPC3 Adnectin further comprises a C-terminal module selected from _{PmXn , PmCXn ,} and _PmCXn1CXn2 , wherein X is any amino acid, and m, n, _n1 , and n2 are independently 0 or an integer of 1, 2, 3, 4, ₅ , or greater.

In some embodiments, the anti-GPC3 Adnectin comprises an amino acid sequence selected from 5, 9-18, 22-31, 35-44, 48-57, 61-70, 74-83, 87-98, 102-128, 130-155, 157-182, 184-209, 211-236, 238-263, 265-290, 292-317, and 319-343.

In some embodiments, the anti-GPC3 Adnectin comprises an amino acid sequence selected from SEQ ID NO: 98, 102-128, 129-155, 157-182, 184-209, 211-236, 238-263, 265-290, 292-317, and 319-343, and optionally has one or more histidines (e.g., 6xHis) at the C-terminus.

In certain embodiments, the anti-GPC3 Adnectin comprises an amino acid sequence selected from SEQ ID NOs: 102-127. In certain embodiments, the anti-GPC3 Adnectin comprises SEQ ID NOs: 114-118. In other embodiments, the anti-GPC3 Adnectin comprises SEQ ID NOs: 123-127.

Provided herein ^are polypeptides that specifically bind to human GPC3 with a KD of ^10-7 or less, wherein the polypeptide comprises ^a10Fn3 domain comprising the BC, DE, and FG loops of ADX_6077_A01, i.e., SEQ ID NOs: 99, 100, and 101. Provided herein are polypeptides that specifically bind to human GPC3 with a KD of ^10-7 or less, wherein the ^polypeptide comprises ^a10Fn3 domain having a BC loop comprising SEQ ID NO: 99, a DE loop comprising SEQ ID NO: 100, and a FG loop comprising SEQ ID NO: 101. Also provided is a polypeptide that specifically binds to human GPC3 with a KD of ^10-7 or less, wherein the polypeptide comprises ^a10Fn3 domain having a BC loop comprising SEQ ID NO: ⁹⁹ , a DE loop comprising SEQ ID NO:100, and a FG loop comprising SEQ ID NO:101, wherein one of the two amino acid residues DG in the FG loop is substituted with another amino acid. Also provided is a polypeptide that specifically binds to human GPC3 with a KD of ^10-7 or less, wherein the polypeptide comprises ^a10Fn3 domain having a BC loop comprising SEQ ID NO:99, a ^DE loop comprising SEQ ID NO:100, and a FG loop comprising SEQ ID NO:101, wherein the amino acid residue D (i.e., D78; numbered relative to the numbering in SEQ ID NO:102) of DG in the FG loop is substituted with another amino acid, such as E, S, A, and G. Also provided are polypeptides that specifically bind to human GPC3 with a KD of ^10-7 or less, wherein the polypeptide comprises ^a10Fn3 domain having a BC loop comprising SEQ ID NO: ⁹⁹ , a DE loop comprising SEQ ID NO:100, and a FG loop comprising SEQ ID NO:101, wherein the amino acid residue G of DG in the FG loop (i.e., D79) is substituted with another amino acid residue, such as S, A, L, or V. Also provided are polypeptides that specifically bind to human GPC3 with a KD of ^10-7 or less, wherein the polypeptide comprises ^a10Fn3 domain having a BC loop comprising SEQ ID NO:99, a DE loop comprising SEQ ID NO:100, and a FG loop comprising SEQ ID NO:101, 129, 156, 183, 210, 237, ²⁶⁴ , 291, or 318. Any of the polypeptides described in this paragraph may comprise a cysteine residue linked directly or indirectly to the C-terminus of the polypeptide and/or may comprise one of the following amino acid residues or sequences linked directly or indirectly to the C-terminus of the polypeptide: P, PC, PCHHHHHH (SEQ ID NO: 395), PCPPPPPC (SEQ ID NO: 416), or PCPPPPPCHHHHHH (SEQ ID NO: 424).

Also provided are polypeptides that specifically bind to human GPC3 with a KD of ^10-7 or less, wherein the polypeptide comprises ^a10Fn3 domain comprising the ^ADX_6077_A01 core sequence (i.e., SEQ ID NO:98) or an amino acid sequence that is at least 90%, 95%, 97%, 98% or 99% identical thereto or differs therefrom by 1-10, 1-5, 1-3, 1-2 or 1 amino acid substitution (e.g., conservative amino acid substitution), deletion or addition. Also provided are polypeptides that specifically bind to human GPC3 with a KD of ^10-7 or less, wherein the polypeptide comprises ^a10Fn3 domain comprising the ^ADX_6077_A01 core sequence (i.e., SEQ ID NO:98) or an amino acid sequence that is at least 90%, 95%, 97%, 98% or 99% identical thereto or differs therefrom by 1-10, 1-5, 1-3, 1-2 or 1 amino acid substitutions (e.g., conservative amino acid substitutions), deletions or additions, and ^the10Fn3 domain has a BC loop comprising SEQ ID NO:99, a DE loop comprising SEQ ID NO:100, and a FG loop comprising SEQ NO:101 (or differs therefrom by one amino acid in DG). Also provided is a polypeptide that specifically binds to human GPC3 with a KD of ^10-7 or less, wherein the polypeptide comprises ^a10Fn3 domain comprising SEQ ID NO:98, or an amino acid sequence that is at least 90%, 95%, 97%, 98% or 99% identical thereto, or that differs therefrom by 1-10, 1-5, 1-3, 1-2 or ¹ amino acid substitutions (e.g., conservative amino acid substitutions), deletions or additions, and ^the10Fn3 domain has a BC loop comprising SEQ ID NO:99, a DE loop comprising SEQ ID NO:100, and a FG loop comprising SEQ ID NO:101, 129, 156, 183, 210, 237, 264, 291 or 318. Also provided are polypeptides that specifically bind to human GPC3 with a KD of ^10-7 or less, wherein the polypeptide comprises ^a10Fn3 domain comprising the ADX_6077_A01 core sequence (i.e., SEQ ID NO: 98) or an amino acid sequence that is at least 90%, 95%, 97%, 98% or 99% identical thereto or differs therefrom by 1-10, 1-5, 1-3 ^, 1-2 or 1 amino acid substitutions (e.g., conservative amino acid substitutions), deletions or additions, and further comprises a cysteine residue directly or indirectly linked to the C-terminus of the polypeptide. Also provided are polypeptides that specifically bind to human GPC3 with a KD of ^10-7 or less, wherein the polypeptide comprises ^a10Fn3 domain comprising the ^ADX_6077_A01 core sequence (i.e., SEQ ID NO:98) or an amino acid sequence that is at least 90%, 95%, 97%, 98% or 99% identical thereto or differs therefrom by 1-10, 1-5, 1-3, 1-2 or 1 amino acid substitutions (e.g., conservative amino acid substitutions), deletions or additions, and further comprises one of the following amino acid residues or sequences linked directly or indirectly to the C-terminus of the polypeptide: P, PC, PCHHHHHH (SEQ ID NO:395), PCPPPPPC (SEQ ID NO:416), or PCPPPPPCHHHHHH (SEQ ID NO:424).

Provided herein are polypeptides comprising the amino acid sequence of ADX_6077_A01 or ADX_6912_G02 (with or without the N-terminal methionine) and with or without a 6xHis tail.

Also provided herein is ^a10Fn3 protein that specifically binds to human GPC3 with a KD of ^10-7 or less and comprises the BC, DE and FG loops of ADX_6077_A01, i.e., SEQ ID NOs: 99, 100 and 101. Provided herein is ^a10Fn3 protein ^that specifically binds to human GPC3 with a KD of ^10-7 or less and has a BC loop comprising SEQ ID NO: 99, a DE loop comprising SEQ ID NO: 100, and a FG loop comprising SEQ ID NO: 101. Also provided is ^{a10Fn3 protein that} specifically binds to human GPC3 with a KD of ^10-7 or less and has a BC loop comprising SEQ ID NO: 99, a DE loop comprising SEQ ID NO: 100, and a FG loop comprising SEQ ID NO: 101, wherein one of the two amino acid residues DG in the FG loop is substituted with another amino acid. Also provided is ^{a10Fn3 protein} that specifically binds to human GPC3 with a KD of ^10-7 or less and has a BC loop comprising SEQ ID NO:99, a DE loop comprising SEQ ID NO:100, and a FG loop comprising SEQ ID NO:101, wherein the amino acid residue D of DG in the FG loop (i.e., D78; numbered relative to the numbering in SEQ ID NO:102) is substituted with another amino acid, such as E, S, A, and G. Also provided is ^a10Fn3 protein ^that specifically binds to human GPC3 with a KD of ^10-7 or less and has a BC loop comprising SEQ ID NO:99, a DE loop comprising SEQ ID NO:100, and a FG loop comprising SEQ ID NO:101, wherein the amino acid residue G of DG in the FG loop (i.e., D79) is substituted with another amino acid residue, such as S, A, L, or V. Also provided ^{are10Fn3 proteins} that specifically bind to human GPC3 with a KD of ^10-7 or less and have a BC loop comprising SEQ ID NO:99, a DE loop comprising SEQ ID NO:100, and a FG loop comprising SEQ ID NO:101, 129, 156, 183, 210, 237, 264, 291, or 318. ^Any10Fn3 protein described in this paragraph may comprise a cysteine residue linked directly or indirectly to its C-terminus and/or may comprise one of the following amino acid residues or sequences linked directly or indirectly to its C-terminus: P, PC, PCHHHHHH (SEQ ID NO:395), PCPPPPPC (SEQ ID NO:416), or PCPPPPPCHHHHHH (SEQ ID NO:424).

Also provided is ^a10Fn3 protein ^that specifically binds to human GPC3 with a KD of ^10-7 or less and comprises the ADX_6077_A01 core sequence (i.e., SEQ ID NO:98) or an amino acid sequence that is at least 90%, 95%, 97%, 98% or 99% identical thereto or that differs therefrom by 1-10, 1-5, 1-3, 1-2 or 1 amino acid substitution (e.g., conservative amino acid substitution), deletion or addition. Also provided is ^a10Fn3 protein ^that specifically binds to human GPC3 with a KD of ^10-7 or less and comprises the ADX_6077_A01 core sequence (i.e., SEQ ID NO:98) or an amino acid sequence that is at least 90%, 95%, 97%, 98% or 99% identical thereto or differs therefrom by 1-10, 1-5, 1-3, 1-2 or 1 amino acid substitution (e.g., conservative amino acid substitution), deletion or addition, and has a BC loop comprising SEQ ID NO: ⁹⁹ , a DE loop comprising SEQ ID NO:100, and a FG loop comprising SEQ NO:101 (or differs therefrom by one amino acid in DG). Also provided is ^a10Fn3 protein ^that specifically binds to human GPC3 with a KD of ^10-7 or less and comprises SEQ ID NO:98, or an amino acid sequence that is at least 90%, 95%, 97%, 98% or 99% identical thereto, or differs therefrom by 1-10, 1-5, 1-3, 1-2 or 1 amino acid substitutions (e.g., conservative amino acid substitutions), deletions or additions, and has a BC loop comprising SEQ ID NO:99, a ^DE loop comprising SEQ ID NO:100, and a FG loop comprising SEQ ID NO:101, 129, 156, 183, 210, 237, 264, 291 or 318. Also provided is ^a10Fn3 protein ^that specifically binds to human GPC3 with a KD of ^10-7 or less and comprises ^a10Fn3 domain comprising the ADX_6077_A01 core sequence (i.e., SEQ ID NO:98) or an amino acid sequence that is at least 90%, 95%, 97%, 98% or 99% identical thereto or differs therefrom by 1-10, 1-5, 1-3, ^1-2 or 1 amino acid substitutions (e.g., conservative amino acid substitutions), deletions or additions, and further comprises a cysteine residue directly or indirectly linked to the C-terminus of ^the10Fn3 protein. Also provided is ^a10Fn3 protein ^that specifically binds to human GPC3 with a KD of ^10-7 or less and comprises ^a10Fn3 domain comprising the ADX_6077_A01 core sequence (i.e., SEQ ID NO:98) or an amino acid sequence that is at least 90%, 95%, 97%, 98% or 99% identical thereto or differs therefrom by 1-10, 1-5, 1-3, ^1-2 or 1 amino acid substitutions (e.g., conservative amino acid substitutions), deletions or additions, and further comprises one of the following amino acid residues or sequences linked directly or indirectly to the C-terminus of ^the10Fn3 protein: P, PC, PCHHHHHH (SEQ ID NO:395), PCPPPPPC (SEQ ID NO:416) or PCPPPPPCHHHHHH (SEQ ID NO:424).

Provided herein ^are10Fn3 proteins comprising the amino acid sequence of ADX_6077_A01 or ADX_6912_G02 (with or without the N-terminal methionine) and with or without a 6xHis tail.

Also provided are drug conjugates comprising one of the polypeptides ^or10Fn3 proteins described in the above paragraphs conjugated to a drug moiety, such as a tubulysin analog.

Further provided ^are10Fn3 proteins or polypeptides comprising10Fn3 domains, ^the10Fn3 domains comprising a sequence including one or more cysteines at their C-termini, wherein at least one cysteine is conjugated to a tubulysin analog described herein. For example, ^10Fn3 proteins or polypeptides ^{comprising10Fn3} domains can be linked to a peptide comprising the amino acid sequence PmCn, wherein m and n are independently integers of ¹ or greater, and wherein one or more cysteines are conjugated to a tubulysin analog described herein.

In certain embodiments, the BC, DE and/or FG loop amino acid sequences of any of the anti-GPC3 Adnectins described herein (e.g., SEQ ID NOs: 5, 9-18, 22-31, 35-44, 48-57, 61-70, 74-83, 87-98, 102-128, 130-155, 157-182, 184-209, 211-236, 238-263, 265-290, 292-317 and 319-343) are grafted into a non- ^10Fn3 domain protein scaffold. For example, one or more loop amino acid sequences are exchanged for one or more CDR loops of an antibody heavy chain or light chain or fragment thereof or inserted into one or more CDR loops of an antibody heavy chain or light chain or fragment thereof. In other embodiments, the protein domain in which one or more amino acid loop sequences are exchanged or inserted includes, but is not limited to, a consensus Fn3 domain (Centocor, US), ankyrin repeat proteins (Molecular Partners AG, Zurich Switzerland), domain antibodies (Domantis, Ltd, Cambridge, MA), single-domain camelid nanobodies (Ablynx, Belgium), lipocalins (e.g., anticalins; Pieris Proteolab AG, Freising, Germany), Avimers (Amgen, CA), affibodies (Affibody AG, Sweden), ubiquitins (e.g., affilins; Scil Proteins GmbH, Halle, Germany), protein epitope mimetics (Polyphor Ltd, Allschwil, Switzerland), helical bundle scaffolds (e.g., alpha bodies, Complix, Belgium), Fyn SH3 domains (Covagen AG, Switzerland), or atrimers (Anaphor, Inc., CA).

B. Cross-competing anti-GPC3 Adnectin

Also provided are Adnectins that compete (e.g., cross-compete) with specific anti-GPC3 Adnectins described herein for binding to human GPC3. Such competing Adnectins can be identified based on their ability to competitively inhibit the binding of the Adnectins described herein to GPC3 in standard GPC3 binding assays. For example, a standard ELISA assay can be used in which recombinant GPC3 protein is immobilized on a plate and one of the Adnectins is fluorescently labeled, and the ability of the unlabeled Adnectin to compete away the binding of the labeled Adnectin can be assessed.

In certain embodiments, a competition ELISA format can be performed to determine whether two anti-GPC3 Adnectins bind to overlapping Adnectin binding sites on GPC3. In one format, Adnectin #1 is coated on a plate, which is then blocked and washed. To this plate is added GPC3 alone, or GPC3 pre-incubated with a saturating concentration of Adnectin #2. After an appropriate incubation period, the plate is washed and probed with a polyclonal anti-GPC3 antibody (such as a biotinylated anti-GPC3 polyclonal antibody), followed by detection with a streptavidin-HRP conjugate and a standard tetramethylbenzidine development procedure. If the OD signal is the same for pre-incubation with or without Adnectin #2, the two Adnectins bind independently of each other and their Adnectin binding sites do not overlap. However, if the OD signal of the wells receiving the GPC3/Adnectin #2 mixture is lower than those receiving only GPC3, the binding of Adnectin #2 is confirmed to block the binding of Adnectin #1 to GPC3.

Alternatively, similar experiments are performed by surface plasmon resonance (SPR, e.g., BIAcore). Adnectin #1 is immobilized on the surface of an SPR chip, followed by injection of GPC3 alone or GPC3 pre-incubated with a saturating concentration of Adnectin #2. If the binding signal for the GPC3/Adnectin #2 mixture is the same as or higher than that for GPC3 alone, the two Adnectins bind independently of each other and their Adnectin binding sites do not overlap. However, if the binding signal for the GPC3/Adnectin #2 mixture is lower than that for GPC3 alone, the binding of Adnectin #2 is confirmed to block the binding of Adnectin #1 to GPC3. These experiments are characterized by the use of saturating concentrations of Adnectin #2. If GPC3 is not saturated with Adnectin #2, the above conclusions do not hold. Similar experiments can be used to determine whether any two GPC3 binding proteins bind to overlapping Adnectin binding sites.

The two assays exemplified above can also be performed in the reverse order, with Adnectin #2 being immobilized and GPC3-Adnectin #1 being added to the plate. Alternatively, Adnectin #1 and/or #2 can be replaced with monoclonal antibodies and/or soluble receptor-Fc fusion proteins.

In another embodiment, competition can be determined using an HTRF sandwich assay.

Candidate competing anti-GPC3 Adnectins may inhibit binding of an anti-GPC3 Adnectin described herein to GPC3 by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%, and/or their binding is inhibited by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%. The % of competition may be determined using the methods described above.

In some embodiments, the molecules that compete with the anti-GPC3 Adnectins described herein are not necessarily Adnectins, but can be any type of molecule that binds to GPC3, such as, but not limited to, antibodies, small molecules, peptides, and the like.

In certain embodiments, the Adnectin binds to the same Adnectin binding site on GPC3 as a specific anti-GPC3 Adnectin described herein. Standard mapping techniques, such as protease mapping, mutational analysis, HDX-MS, X-ray crystallography, and 2-dimensional nuclear magnetic resonance can be used to determine whether an Adnectin binds to the same Adnectin binding site as a reference Adnectin (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, ed. (1996)).

In some embodiments, the anti-GPC3 Adnectins provided herein bind to discontinuous Adnectin binding sites on human GPC3. In some embodiments, the anti-GPC3 FBS binds to a region of, e.g., 10-20 amino acid residues within human GPC3 (SEQ ID NO: 344) comprising SEQ ID NO: 345. In some embodiments, the anti-GPC3 Adnectin binds to a region of, e.g., 10-20 amino acid residues within human GPC3 (SEQ ID NO: 344) comprising SEQ ID NO: 346. In other embodiments, the anti-GPC3 FBS binds to two regions of, e.g., 10-20 amino acid residues each within human GPC3 (SEQ ID NO: 344), respectively one region comprising SEQ ID NO: 345 and the other region comprising SEQ ID NO: 346.

C. N-terminally and C-terminally modified anti-GPC3 Adnectin

In some embodiments, the amino acid sequence of the N-terminal and/or C-terminal regions of the Adnectin may be modified by deletion, substitution, or insertion relative to the amino acid sequence of the corresponding region of ^the10Fn3 domain comprising, for example, SEQ ID NO:1.

In certain embodiments, in the polypeptides provided herein, the amino acid sequence of the first 1, 2, 3, 4, 5, 6, 7, 8, or 9 residues of an Adnectin, e.g., a sequence beginning with "VSD" as in SEQ ID NO: 1, can be modified or deleted. In exemplary embodiments, the amino acids corresponding to amino acids 1-7, 8, or 9 of an Adnectin having a sequence beginning with "VSD", e.g., as in SEQ ID NO: 1, are replaced with an alternative N-terminal region having a length of 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, 1-2, or 1 amino acids.

Exemplary alternative N-terminal regions that can be added to the GPC3 Adnectin core sequence or those sequences beginning with "VSD" include (in single letter amino acid code) M, MG, G, MGVSDVPRD (SEQ ID NO: 351), and _GVSDVPRD (SEQ ID NO: 352). Other suitable alternative N-terminal regions include, for example, _XnSDVPRDL (SEQ ID NO: 353), _XnDVPRDL (SEQ ID NO: 354), XnVPRDL (SEQ ID NO: 355), _XnPRDL (SEQ ID NO: 356), _XnRDL (SEQ ID NO: 357), _{XnDL (SEQ ID NO: 358), or XnL ,} wherein n=0, 1 or 2 amino acids, wherein when n=1, X is Met or Gly, and when n=2, X is Met-Gly. When a Met-Gly sequence is added to ¹⁰ When the N-terminus of the Fn3 domain is cleaved, M is often cleaved off, leaving G at the N-terminus. In other embodiments, the alternative N-terminal region comprises the amino acid sequence MASTSG (SEQ ID NO: 359). In certain embodiments, the N-terminal extension consists of an amino acid sequence selected from the group consisting of: M, MG, and G.

In some embodiments, an alternative C-terminal region having a length of 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, 1-2, or 1 amino acids can be added to the C-terminal region of the GPC3 Adnectin that ends with "RT" (as in, for example, SEQ ID NO: 1). Examples of alternative C-terminal region sequences include, for example, polypeptides comprising, consisting essentially of, or consisting of EIEK (SEQ ID NO: 360), EGSGC (SEQ ID NO: 361), EIEKPCQ (SEQ ID NO: 362), EIEKPSQ (SEQ ID NO: 363), EIEKP (SEQ ID NO: 364), EIEKPS (SEQ ID NO: 365), EIEKPC (SEQ ID NO: 366), EIDK (SEQ ID NO: 367), EIDKPCQ (SEQ ID NO: 368), or EIDKPSQ (SEQ ID NO: 369). In certain embodiments, the C-terminal region consists of EIDKPCQ (SEQ ID NO: 368). In certain embodiments, the ¹⁰ Fn3 domain is linked to a C-terminal extension sequence comprising E and D residues and may be between 8 and 50, 10 and 30, 10 and 20, 5 and 10, and 2 and 4 amino acids in length. In some embodiments, the tail sequence comprises an ED-based linker, wherein the sequence comprises tandem repeats of ED. In exemplary embodiments, the tail sequence comprises 2-10, 2-7, 2-5, 3-10, 3-7, 3-5, 3, 4, or 5 ED repeats. In certain embodiments, the ED-based tail sequence may also comprise additional amino acid residues, such as, for example, EI, EID, ES, EC, EGS, and EGC. Such sequences are based in part on known Adnectin tail sequences, such as EIDKPSQ (SEQ ID NO: 369), in which residues D and K have been removed. In some embodiments, the ED-based tail comprises an E, I, or EI residue preceding the ED repeat.

In certain embodiments, the N- or C-terminal extension sequence is linked to ^the10Fn3 domain using a known linker sequence (e.g., SEQ ID NOs: 426-451 in Table 13). In some embodiments, the sequence can be placed at the C-terminus of ^the10Fn3 domain to facilitate attachment of a pharmacokinetic module. For example, a cysteine-containing linker such as GSGC can be added to the C-terminus to facilitate site-directed PEGylation on a cysteine residue.

In certain embodiments, an alternative C-terminal module that can be linked to the C-terminal amino acid RT of a GPC3 Adnectin (i.e., for example, as amino acid 94 in SEQ ID NO: 1 ₎ comprises amino acids _PmXn , wherein P is proline, X is any amino acid, m is an integer of at least 1, and n is 0 or an integer of at least 1. In some embodiments, m can be 1, 2, 3 or greater. For example, m can be 1-3 or m can be 1-2. "n" can be 0, 1, 2, 3 or greater, for example, n can be 1-3 or 1-2.

The _PmXn module can be directly linked to the C-terminal amino acid of ^the10Fn3 module, for example, its 94th amino acid (based on the amino acid numbering of SEQ ID NO: 1). _{The PmXn module} can be linked to the 94th amino acid of ^the10Fn3 module via a peptide bond. The single proline residue at the end of SEQ ID NO: 1 is referred to as "95Pro" or "Pro95" or "P95" or "95P".

In certain embodiments, n is not 0, and can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater. For example, n can be 0-10, 0-5, 0-3, 1-10, 1-5, 1-3 or 1-2. However, more than 10 amino acids can be connected to proline. For example, in a tandem FBS module or an FBS module fused to another polypeptide, the C-terminal amino acid of the FBS module can be connected to one or more prolines, and the last proline is connected to a second FBS module or to a heterologous module. Therefore, in certain embodiments, n can be an integer ranging from 0-100, 0-200, 0-300, 0-400, 0-500 or greater.

In certain embodiments, _{the PmXn} linked to the C-terminus of the GPC3 Adnectin comprises a cysteine. For example, the first amino acid after the proline can be a cysteine, and the cysteine can be the last amino acid in the molecule or the cysteine can be after one or more amino acids. The presence of cysteine allows a heterologous module such as a chemical module (e.g., _PEG ) to be conjugated to the FBS module. Exemplary _{PmXn modules} comprising cysteine include: _PmCXn , wherein C is cysteine, each X is independently any amino acid, m is an integer of at least 1, and n is an integer of 0 or at least 1. In some embodiments, m can be 1, 2, 3 or greater. For example, m can be 1-3 or m can be 1-2. "n" can be 0, 1, 2, 3 or greater, for example, n can be 1-3 or _1-2 . Other exemplary PmXn modules include two cysteines, for example, _PmCXn1CXn2 , wherein each X is independently any amino acid, _n1 and _n2 are independently 0 or an integer of at least 1. For example, _n1 can be 1, 2, 3, 4, or 5, and _n2 can be 1, 2, 3, 4, or 5. Exemplary PmXn modules include those listed in Table 1.

Table 1: Exemplary PmXn modules

In certain embodiments, for example, the PmXn module is selected from PC, PPC, and PCC. In another embodiment, the PmXn module is _PmCXn1CXn2 . In certain embodiments, _PmCXn1CXn2 is selected from _PCPPPC (SEQ ID NO: ₄₁₅ ) and PCPPPPPC (SEQ ID NO: 416).

Any of the C-terminal modifications described herein can be applied to GPC3 Adnectins.

Any PmXn module, for example, those shown in Table 1, may be followed by a histidine tail, for example, a 6xHis tag, or other tag. This does not exclude the possibility of a histidine tail being included in the PmXn.

In certain embodiments, a fibronectin-based scaffold protein comprises ^a10Fn3 domain with both an alternative N-terminal region sequence and an alternative C-terminal region sequence, and optionally a 6X his tail.

II. Multivalent Peptides

In certain embodiments, the protein comprises a GPC3 FBS and at least one other FBS. A multivalent FBS may comprise 2, 3 or more FBSs covalently bound. In an exemplary embodiment, the FBS module is a bispecific or dimeric protein comprising ^two10Fn3 domains.

In the FBS module in the multivalent protein, for example, the ¹⁰ Fn3 domain can be connected by a polypeptide linker. Exemplary polypeptide linkers include polypeptides with 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3 or 1-2 amino acids. Suitable linkers for connecting the ¹⁰ Fn3 domain are those linkers that allow the separated domains to fold independently of each other to form a three-dimensional structure that allows high-affinity binding to the target molecule. Specific examples of suitable linkers include linkers based on glycine-serine, linkers based on glycine-proline, linkers based on proline-alanine, and any other linkers described herein. In some embodiments, the linker is a linker based on glycine-proline. These linkers contain glycine and proline residues and the length can be between 3 and 30, 10 and 30, and 3 and 20 amino acids. Examples of such linkers include GPG, GPGGPPG (SEQ ID NO: 436) and GPGGPGPGPPG (SEQ ID NO: 437). In some embodiments, the linker is a proline-alanine based linker. These linkers contain proline and alanine residues and can be between 3 and 30, 10 and 30, 3 and 20, and 6 and 18 amino acids in length. Examples of such linkers include PAPAPA (SEQ ID NO: 438), PAPAPAPAPAPA (SEQ ID NO: 439), and PAPAPAPAPAPAPAPAPA (SEQ ID NO: 440). In some embodiments, the linker is a glycine-serine based linker. These linkers contain glycine and serine residues and can be between 8 and 50, 10 and 30, and 10 and 20 amino acids in length. Examples of such linkers can include, for example, (GS) _5-10 (SEQ ID NO: 464), (G ₄ S) _2-5 (SEQ ID NO: 465), and (G ₄ S) ₂ G (SEQ ID NO: 466). Examples of such linkers include SEQ ID NO: 427-439. In an exemplary embodiment, the linker does not contain any Asp-Lys(DK) pairs.

III. Pharmacokinetics Module

For therapeutic purposes, the anti-GPC3 Adnectin described herein may be directly or indirectly linked to a pharmacokinetic (PK) module. Improvements in pharmacokinetic can be assessed based on perceived therapeutic needs. It is often desirable to increase bioavailability and/or increase the time between doses, which may be achieved by increasing the time that the protein remains effective in serum after administration. In some cases, it is desirable to improve the continuity of serum concentrations of the protein over time (e.g., reducing the difference in serum concentrations of the protein shortly after administration and before the next administration). Anti-GPC3 Adnectins may be attached to a module that reduces the clearance of the polypeptide in a mammal (e.g., mouse, rat, or human) by more than two-fold, more than three-fold, more than four-fold, or more than five-fold relative to unmodified anti-GPC3 Adnectins. Other measures of pharmacokinetic improvement may include serum half-life, which is generally divided into α and β phases. Either or both phases may be significantly improved by adding appropriate modules. For example, the PK moiety can increase the serum half-life of a polypeptide by more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%, 400%, 600%, 800%, 1000% or more relative to the Fn3 domain alone.

Moieties that slow the clearance of proteins from the blood (referred to herein as "PK moieties") include polyoxyalkylene moieties (e.g., polyethylene glycol), sugars (e.g., sialic acid), and well-tolerated protein moieties (e.g., Fc and fragments and variants thereof, transferrin, or serum albumin). Anti-GPC3 Adnectins may also be fused to albumin or a fragment (portion) or variant of albumin, as described in U.S. Publication No. 2007/0048282, or may be fused to one or more serum albumin binding Adnectins as described herein.

Other PK modules that can be used in the present invention include those described by Kontermann et al. (Current Opinion in Biotechnology 2011; 22: 868-76), which are incorporated herein by reference. Such PK modules include, but are not limited to, human serum albumin fusions, human serum albumin conjugates, human serum albumin binders (e.g., Adnecti PKE, AlbudAb, ABD), XTEN fusions, PAS fusions (i.e., recombinant PEG mimetics based on three amino acids: proline, alanine, and serine), carbohydrate conjugates (e.g., hydroxyethyl starch (HES)), glycosylation, polysialic acid conjugates, and fatty acid conjugates.

In some embodiments, the invention provides anti-GPC3 Adnectins fused to a PK moiety that is a polymeric carbohydrate. In some embodiments, the PK moiety is a polyethylene glycol moiety or an Fc region. In some embodiments, the PK moiety is a serum albumin binding protein, such as those described in U.S. Publication Nos. 2007/0178082 and 2007/0269422. In some embodiments, the PK moiety is human serum albumin. In some embodiments, the PK moiety is transferrin.

In some embodiments, the PK module is connected to the anti-GPC3 Adnectin via a polypeptide linker. Exemplary polypeptide linkers include polypeptides having 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, or 1-2 amino acids. Suitable linkers for connecting Fn3 domains are those that allow separate domains to fold independently of each other to form a three-dimensional structure that allows high affinity binding to the target molecule. In an exemplary embodiment, the linker does not contain any Asp-Lys (DK) pairs. A list of suitable linkers is provided in Table 14 (e.g., SEQ ID NOs: 426-451).

In some embodiments, the anti-GPC3 Adnectin is linked to the anti-HSA Adnectin, for example, via a polypeptide linker having a protease site that can be cleaved by proteases in the blood or target tissue. Such embodiments can be used to release the anti-GPC3 Adnectin for better delivery or therapeutic properties or more efficient production.

An additional linker or spacer may be introduced at the N-terminus or C-terminus of the Fn3 domain so that it is located between the Fn3 domain and the polypeptide linker.

Polyethylene glycol

In some embodiments, the anti-GPC3 Adnectin comprises polyethylene glycol (PEG). PEG is a well-known water-soluble polymer that is commercially available or can be prepared by ring-opening polymerization of ethylene glycol according to methods well known in the art (Sandler and Karo, Polymer Synthesis, Academic Press, New York, Volume 3, pages 138-161). The term "PEG" is broadly used to cover any polyethylene glycol molecule, regardless of the size or terminal modification of the PEG, and can be represented by the following formula: X—O(CH ₂ CH ₂ O) _n-1 CH ₂ CH ₂ OH, where n is 20 to 2300, X is H or a terminal modification, such as a C _1-4 alkyl. PEG may contain other chemical groups that are necessary for the binding reaction, which are generated by the chemical synthesis of the molecule; or, it is used as a spacer for the optimal distance of multiple parts of the molecule. In addition, such PEG can be composed of one or more PEG side chains connected together. PEGs with more than one PEG chain are called multi-arm or branched PEGs. Branched PEGs are described, for example, in European Published Application No. 473084A and US Pat. No. 5,932,462.

Immunoglobulin Fc domain (and fragments)

In certain embodiments, the anti-GPC3 Adnectin is fused to an immunoglobulin Fc domain or a fragment or variant thereof. As used herein, a "functional Fc region" is an Fc domain or fragment thereof that retains the ability to bind to FcRn. In some embodiments, a functional Fc region binds to FcRn but does not have effector function. The ability of an Fc region or fragment thereof to bind to FcRn can be determined by standard binding assays known in the art. In other embodiments, an Fc region or fragment thereof binds to FcRn and has at least one "effector function" of a native Fc region. Exemplary "effector functions" include C1q binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions generally require the Fc region to bind to a binding domain (e.g., an anti-GPC3 Adnectin) and can be assessed using various assays known in the art for evaluating such antibody effector functions.

A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. A "variant Fc region" comprises an amino acid sequence that differs from a native sequence Fc region due to at least one amino acid modification. Preferably, the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or to the Fc region of a parent polypeptide, for example, from about one to about ten amino acid substitutions in the native sequence Fc region or in the Fc region of a parent polypeptide, and preferably from about one to about five amino acid substitutions. The variant Fc region herein will preferably have at least about 80% sequence identity with the native sequence Fc region and/or with the Fc region of a parent polypeptide, and most preferably at least about 90% sequence identity therewith, more preferably at least about 95% sequence identity therewith.

In an exemplary embodiment, the Fc domain is derived from the IgG1 subclass, however, other subclasses (e.g., IgG2, IgG3, and IgG4) may also be used. Shown below is the sequence of a human IgG1 immunoglobulin Fc domain:

DKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:463)

The core hinge sequence is underlined, and the CH2 and CH3 regions are represented in conventional text. It should be understood that the C-terminal lysine is optional. Allotypes and mutants of this sequence may also be used. As is known in the art, mutants may be designed to modulate various properties of Fc, such as ADCC, CDC or half-life.

In certain embodiments, the Fc region for anti-GPC3 Adnectin fusions comprises a CH1 region. In certain embodiments, the Fc region for anti-GPC3 Adnectin fusions comprises a CH2 and CH3 region. In certain embodiments, the Fc region for anti-GPC3 Adnectin fusions comprises a CH2, CH3, and hinge region.

In certain embodiments, the "hinge" region comprises the core hinge residues of positions 1-16 of SEQ ID NO: 463 (DKTHTCPPCPAPELLG; SEQ ID NO: 464) spanning the IgG1 Fc region. In certain embodiments, the anti-GPC3 Adnectin-Fc fusion adopts a multimeric structure (e.g., a dimer) due in part to the cysteine residues at positions 6 and 9 of SEQ ID NO: within the hinge region.

IV. Vectors and Polynucleotides

Also provided herein are nucleic acids encoding the anti-GPC3 Andectins described herein. As will be appreciated by those skilled in the art, due to the third base degeneracy, almost every amino acid can be represented by more than one triplet codon in the encoding nucleotide sequence. In addition, minor base pair changes may result in conservative substitutions in the encoded amino acid sequence, but are not expected to substantially change the biological activity of the gene product. Therefore, the nucleic acid sequence encoding the protein described herein may be slightly modified in sequence while still encoding its corresponding gene product. Certain exemplary nucleic acids encoding the anti-GPC3 Adnectins and fusions thereof described herein include nucleic acids having sequences listed in SEQ ID NOs: 452-462.

Nucleic acids encoding any of the various proteins disclosed herein, including anti-GPC3 Adnectins, can be synthesized chemically, enzymatically, or recombinantly. Codon usage can be selected to improve expression in cells. Such codon usage will depend on the cell type selected. Codon usage patterns specifically for E. coli and other bacteria, as well as mammalian cells, plant cells, yeast cells, and insect cells have been developed. See, e.g., Mayfield et al., Proc. Natl. Acad. Sci. USA, 100(2):438-442 (Jan. 21, 2003); Sinclair et al., Protein Expr. Purif., 26(1):96-105 (Oct. 2002); Connell, N.D., Curr. Opin. Biotechnol., 12(5):446-449 (Oct. 2001); Makrides et al., Microbiol. Rev., 60(3):512-538 (Sep. 1996); and Sharp et al., Yeast, 7(7):657-678 (Oct. 1991).

The general techniques of nucleic acid manipulation are described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Vols. 1-3, Cold Spring Harbor Laboratory Press (1989), or Ausubel, F. et al., Current Protocols in Molecular Biology, Green Publishing and Wiley-Interscience, New York (1987) and regular updates, which are incorporated herein by reference. The DNA encoding the polypeptide is operably linked to a transcription or translation regulatory element suitable for deriving from a mammalian, viral, or insect gene. Such regulatory elements include a transcription promoter, an optional operating sequence for controlling transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for controlling the termination of transcription and translation. In addition, the ability to replicate in a host, which is often conferred by a replication origin, and a selection gene that is convenient for identifying transformants are incorporated.

The proteins described herein can be produced not only directly in a recombinant manner, but also in the form of polypeptides with heterologous polypeptides, preferably signal sequences or other polypeptides with specific cleavage sites at the N-terminus of mature proteins or polypeptides. The heterologous signal sequence preferably selected is a signal sequence that is recognized and processed by the host cell (i.e., cleaved by a signal peptidase). For prokaryotic host cells that do not recognize and process natural signal sequences, the signal sequence is replaced by a prokaryotic signal sequence selected from the group consisting of, for example, alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion, the natural signal sequence can be replaced by, for example, a yeast invertase leader, an alpha factor leader (including saccharomyces and Kluyveromyces alpha-factor leaders), or an acid phosphatase leader, a Candida albicans glucoamylase leader, or a signal described in PCT Publication No. WO 90/13646. In mammalian cell expression, mammalian signal sequences and viral secretory leaders, such as herpes simplex gD signals, can be used. Such a precursor region DNA may be linked in reading frame to the protein encoding DNA.

Both expression vector and cloning vector comprise the nucleotide sequence that enables the vector to replicate in one or more selected host cells.Usually, in cloning vector, this sequence is the sequence that enables the vector to replicate independently of host chromosome DNA, and comprises replication origin or autonomous replication sequence.Such sequence for various bacteria, yeast and virus is well known.The replication origin from plasmid pBR322 is suitable for most gram-negative bacteria, 2 μ plasmid starting point is suitable for yeast, and various virus starting points (SV40, polyoma virus, adenovirus, VSV or BPV) can be used for cloning vector in mammalian cell.Usually, the replication origin component is unnecessary for mammalian expression vector (generally SV40 starting point can be used, only because it contains early promoter).

Expression vectors and cloning vectors may contain a selection gene, also known as a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins such as ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) provide key nutrients not available in complex media, e.g., the gene encoding D-alanine racemase of Bacillus.

A suitable selection gene for use in yeast is the trp1 gene, which is present in the yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). The trp1 gene targets a yeast mutant lacking the ability to grow in tryptophan, e.g. No. 44076 or PEP4-1 provide selection markers. Jones, Genetics, 85: 12 (1977). In addition, the presence of trp1 lesions in the yeast host cell genome provides an effective environment for detecting transformation by growth in the absence of tryptophan. Similarly, Leu2-deficient yeast strains ( 20,622 or 38,626) were complemented by a known plasmid carrying the Leu2 gene.

Expression vectors and cloning vectors often contain promoters that are recognized by the host organism and are operably connected to the nucleic acid encoding the protein. Promoters suitable for use with prokaryotic hosts include phoA promoters, beta-lactamase and lactose promoter systems, alkaline phosphatase, tryptophan (trp) promoter systems, and hybrid promoters such as tac promoters. However, other known bacterial promoters are also suitable. Promoters used in bacterial systems will also include Shine-Dalgarno (S.D.) sequences, which are operably connected to the DNA encoding the protein.

The promoter sequence of eukaryotic organisms is known.Almost all eukaryotic genes have an AT-rich region, which is located about 25 to 30 bases upstream of the site where transcription starts.Another sequence found 70 to 80 bases upstream of the transcription start site of many genes is a CNCAAT (SEQ ID NO:465) region, in which N can be any nucleotide.At the 3' end of most eukaryotic genes is an AATAAA (SEQ ID NO:466) sequence, which can be a signal that adds a poly A tail to the 3' end of the coding sequence.All of these sequences are suitably inserted into eukaryotic expression vectors.

Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, 3-phosphoglyceraldehyde dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, 6-phosphoglucose isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters with the additional advantage of controlling transcription by growth conditions, are the promoter regions for the following enzymes: alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothioneins, 3-phosphate glyceraldehyde dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in European Patent Publication No. 73,657 and PCT Publication Nos. WO 2011/124718 and WO 2012/059486. It is also advantageous to use a yeast enhancer with a yeast promoter.

Transcription from the vector in a mammalian host cell can be driven, for example, by promoters obtained from viral genomes, promoters from heterologous mammalian cells, such as The promoter may be controlled by a promoter or an immunoglobulin promoter, a heat shock promoter, as long as such promoter is compatible with the host cell system, such as polyoma virus, fowl pox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and most preferably, simian virus 40 (SV40).

The early and late promoters of the SV40 virus are conveniently obtained in the form of an SV40 restriction fragment, which also contains the SV40 viral replication origin. The immediate early promoter of the human cytomegalovirus is conveniently obtained in the form of a HindIII E restriction fragment. A system for expressing DNA in a mammalian host using bovine papilloma virus as a vector is disclosed in U.S. Patent No. 4,419,446. Modifications to this system are described in U.S. Patent No. 4,601,978. Regarding the expression of human beta interferon cDNA in mouse cells under the control of a thymidine kinase promoter from herpes simplex virus, see also Reyes et al., Nature, 297: 598-601 (1982). Alternatively, the Rous sarcoma virus long terminal repeat sequence can be used as a promoter.

The transcription of protein-coding DNA by higher eukaryotes is often increased by inserting enhancer sequences into the vector. Currently, many enhancer sequences from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin) are known. However, enhancers from eukaryotic cell viruses are usually used. Examples include the SV40 enhancer (bp100-270) located downstream of the replication origin (late side), the cytomegalovirus early promoter enhancer, the polyoma enhancer located downstream of the replication origin (late side), and the adenovirus enhancer. Regarding the enhancing elements for eukaryotic promoter activation, see also Yaniv, Nature, 297: 17-18 (1982). The enhancer can be spliced into the vector at the 5' or 3' position of the polypeptide coding sequence but preferably at the 5' site of the promoter.

Expression vectors used in eukaryotic host cells (e.g., yeast, fungi, insects, plants, animals, humans, or nucleated cells from other multicellular organisms) will also contain sequences necessary for terminating transcription and stabilizing the mRNA. Such sequences are commonly available from the 5' and sometimes 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments that are transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the polypeptide. A useful transcription termination component is the bovine growth hormone polyadenylation region. See WO 94/11026 and the expression vectors disclosed therein.

The recombinant DNA may also include any type of protein tag sequence that can be used to purify the protein. Examples of protein tags include, but are not limited to, histidine tags, Tag, myc tag, HA tag, or GST tag. Suitable cloning and expression vectors for bacterial, fungal, yeast, and mammalian cell hosts can be found in: Cloning Vectors: A Laboratory Manual, Elsevier, New York (1985), the relevant disclosure of which is incorporated herein by reference.

The expression construct can be introduced into the host cell using a method appropriate for the host cell, which will be apparent to those skilled in the art. Various methods for introducing nucleic acids into host cells are known in the art, including, but not limited to, electroporation; transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or other substances; microprojectile bombardment; liposome transfection; and infection (where the vector is the infectious agent).

Suitable host cells include prokaryotes, yeast, mammalian cells or bacterial cells. Suitable bacteria include gram-negative or gram-positive organisms, for example, Escherichia coli or Bacillus species. Preferably, yeast from Saccharomyces species, such as Saccharomyces cerevisiae, can also be used to produce polypeptides. Various mammalian or insect cell culture systems can also be used to express recombinant proteins. The baculovirus system for producing heterologous proteins in insect cells is reviewed by Luckow et al. (Bio/Technology, 6:47 (1988)). Examples of suitable mammalian host cell lines include endothelial cells, COS-7 monkey kidney cells, CV-1, L cells, C127, 3T3, Chinese hamster ovary (CHO), human embryonic kidney cells, HeLa, 293, 293T and BHK cell lines. Purified proteins are prepared by culturing a suitable host/vector system to express recombinant proteins. The FBS protein is then purified from culture medium or cell extracts.

V. Protein Production

Host cells are transformed with the expression or cloning vectors described herein for protein production and cultured in a conventional nutrient medium modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequence.

Host cells for protein production can be cultured in a variety of culture media. Commercially available culture media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma)), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM) (Sigma)) are suitable for culturing host cells. In addition, any of the culture media described in Ham et al., Meth. Enzymol., 58: 44 (1979), Barnes et al., Anal. Biochem., 102: 255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; PCT Publication Nos. WO 90/03430; WO 87/00195; or U.S. Pat. No. RE30,985 can be used as a culture medium for host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as gentamicin drugs), trace elements (defined as inorganic compounds, usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements known to those skilled in the art may also be included at appropriate concentrations. The culture conditions, such as temperature, pH, and the like, are those previously used for the host cell selected for expression and will be apparent to the skilled artisan.

Cell-free translation systems can also be used to produce proteins disclosed herein. For such purposes, the nucleic acid encoding the protein must be modified to allow in vitro transcription to produce mRNA and to allow cell-free translation of the mRNA in the specific cell-free system used (eukaryotic cell-free translation system such as mammalian or yeast cell-free translation system, prokaryotic cell-free translation system such as bacterial cell-free translation system).

Proteins may also be produced by chemical synthesis (eg, by the methods described in Solid Phase Peptide Synthesis, 2nd Edition, The Pierce Chemical Co., Rockford, IL (1984)). Modifications of proteins may also be produced by chemical synthesis.

The proteins disclosed herein can be purified by methods commonly known in the field of protein chemistry for separation/purification of proteins. Non-limiting examples include extraction, recrystallization, salting out (e.g., using ammonium sulfate or sodium sulfate), centrifugation, dialysis, ultrafiltration, adsorption chromatography, ion exchange chromatography, hydrophobic chromatography, normal phase chromatography, reverse phase chromatography, gel filtration, gel permeation chromatography, affinity chromatography, electrophoresis, countercurrent distribution, or any combination of these methods. After purification, the protein can be exchanged into different buffers and/or concentrated by any method known in the art, including, but not limited to, filtration and dialysis.

A purified protein is preferably at least 85% pure, more preferably at least 95% pure, and most preferably at least 98% or 99% pure. Regardless of the exact numerical value of purity, the protein is sufficiently pure for use as a pharmaceutical product.

A method for expressing Adnectin in E. coli is as follows. The clone encoding Adnectin is cloned into the pET9d vector, upstream of the HIS ₆ tag, and transformed into E. coli BL21DE3plysS cells, incubated in 5 ml of LB medium containing 50 μg/mL kanamycin in a 24-well format, and cultured overnight at 37°C. Prepare a fresh 5 ml LB medium (50 μg/mL kanamycin) culture for inducing expression by aspirating 200 μl of the overnight culture and distribute it to the appropriate wells. Grow the culture at 37°C until _A600 is 0.6-0.9. After induction with 1 mM isopropyl-β-thiogalactoside (IPTG), the culture is expressed at 30°C for 6 hours and harvested by centrifugation at 2750g for 10 minutes at 4°C.

Cell pellets (24-well format) were lysed by resuspending in 450 μl of lysis buffer (50 mM NaH ₂ PO ₄ , 0.5 M NaCl, 1× Complete ^™ Protease Inhibitor Cocktail-EDTA free (Roche), 1 mM PMSF, 10 mM CHAPS, 40 mM imidazole, 1 mg/ml lysozyme, 30 μg/ml DNase, 2 μg/ml aprotinin, pH 8.0) and shaking at room temperature for 1-3 hours. Lysates were clarified and re-racked to a 96-well format by transfer to a 96-well Whatman GF/D Unifilter equipped with a 96-well 1.2 ml catch plate and filtered with positive pressure. The clarified lysate was transferred to a 96-well nickel-cobalt chelate plate equilibrated with equilibration buffer (50 mM NaH ₂ PO ₄ , 0.5 M NaCl, 40 mM imidazole, pH 8.0) and incubated for 5 minutes. Unbound material was removed with the aid of positive pressure. The resin was washed twice with 0.3 ml/well of wash buffer #1 (50 mM NaH ₂ PO ₄ , 0.5 M NaCl, 5 mM CHAPS, 40 mM imidazole, pH 8.0). Each wash was removed with the aid of positive pressure. Prior to elution, each well was washed with 50 μl of elution buffer (PBS + 20 mM EDTA), incubated for 5 minutes, and this wash was discarded with the aid of positive pressure. Proteins were eluted by applying an additional 100 μl of elution buffer to each well. After incubation at room temperature for 30 minutes, the plate was centrifuged at 200 g for 5 minutes and the eluted protein was collected in a 96-well capture plate containing 5 μl 0.5 M _MgCl2 added to the bottom of the elution capture plate prior to elution. The eluted protein was quantified using a total protein assay using the wild ^type10Fn3 domain as a protein standard.

The medium-scale expression and purification method of insoluble Adnectin is described as follows. The nucleic acid encoding Adnectin is cloned into the pET9d (EMD Bioscience, San Diego, CA) vector together with the HIS ₆ tag and expressed in Escherichia coli HMS174 cells. Use 20ml inoculation culture (generated from a single plate inoculated colony) to inoculate 1 liter of LB medium containing 50μg/ml carbenicillin and 34μg/ml chloramphenicol. The culture is grown at 37°C until _A600 is 0.6-1.0. After induction with 1mM isopropyl-β-thiogalactoside (IPTG), the culture is grown at 30°C for 4 hours and the culture is harvested by centrifugation at 4°C for 30 minutes at>10,000g. The cell pellet is frozen at -80°C. Use The cell pellet was resuspended in 25 ml lysis buffer (20 mM NaH ₂ PO ₄ , 0.5 M NaCl, 1x Complete Protease Inhibitor Cocktail-EDTA free (Roche), 1 mM PMSF, pH 7.4) on ice using a homogenizer (IKAworks). (Microfluidics) high pressure homogenization (> 18,000psi) to achieve cell lysis. The insoluble part was separated by centrifugation at 23,300g for 30 minutes at 4 ° C. The insoluble precipitate recovered from the centrifugation of the lysate was washed with 20mM sodium phosphate/500mM NaCl (pH 7.4). The precipitate was redissolved in 6.0M guanidine hydrochloride in 20mM sodium phosphate/500mM NaCl (pH 7.4) with ultrasonic treatment, and then incubated for 1-2 hours at 37 degrees. The redissolved precipitate was filtered to 0.45 μm and loaded onto a Histrap column balanced with 20mM sodium phosphate/500M NaCl/6.0M guanidine (pH 7.4) buffer. After loading, the column was washed with another 25 CV of the same buffer. Bound protein was eluted with 50 mM imidazole in 20 mM sodium phosphate/500 mM NaCl/6.0 M guanidine-HCl, pH 7.4. The purified protein was refolded by dialysis against 50 mM sodium acetate/150 mM NaCl, pH 4.5.

The medium-scale expression and purification method of soluble Adnectin is described as follows. The nucleic acid encoding Adnectin is cloned into the pET9d (EMD Bioscience, San Diego, CA) vector together with the HIS ₆ tag and expressed in Escherichia coli HMS174 cells. Use 20ml inoculation culture (generated from a single plate inoculated colony) to inoculate 1 liter of LB medium containing 50μg/ml carbenicillin and 34μg/ml chloramphenicol. The culture is grown at 37°C until _A600 is 0.6-1.0. After induction with 1mM isopropyl-β-thiogalactoside (IPTG), the culture is grown at 30°C for 4 hours and the culture is harvested by centrifugation at 4°C for 30 minutes at>10,000g. The cell pellet is frozen at -80°C. Use The cell pellet was resuspended in 25 ml lysis buffer (20 mM NaH ₂ PO ₂ , 0.5 M NaCl, 1x Complete Protease Inhibitor Cocktail-EDTA free (Roche), 1 mM PMSF, pH 7.4) on ice using a homogenizer (IKAworks). (Microfluidics) carries out high pressure homogenization (>18,000psi) to realize cell lysis.Soluble part is separated by centrifugation at 23,300g for 30 minutes at 4 ℃.Supernatant is clarified via 0.45 μm filter.The clarified lysate is loaded onto a Histrap column (GE) pre-equilibrated with 20mM sodium phosphate/500M NaCl (pH7.4).Then the column is washed with 20mM sodium phosphate/500mM NaCl/25mM imidazole (pH7.4) of 25 column volumes of the same buffer, 20 column volumes of 20mM sodium phosphate/500mM NaCl/40mM imidazole (pH 7.4) and 35 column volumes of 20mM sodium phosphate/500M NaCl/40mM imidazole (pH 7.4) subsequently. The protein was eluted with 15 column volumes of 20 mM sodium phosphate/500 mM NaCl/500 mM imidazole, pH 7.4, and fractions were pooled according to absorbance at A280 and dialyzed against 1 x PBS, 50 mM Tris, 150 mM NaCl, pH 8.5, or 50 mM NaOAc, 150 mM NaCl, pH 4.5. Any precipitate was removed by filtration at 0.22 μm.

VI. Biophysical and Biochemical Characterization

The binding of the anti-GPC3 Adnectins described herein can be evaluated in terms of equilibrium constants (e.g., dissociation, _KD ) and in terms of kinetic constants (e.g., association rate constant _kon and dissociation rate constant koff ₎ . Adnectins typically bind to target molecules with a _KD of less than 1 μM, 500 nM, 100 nM, 10 nM, 1 nM, 500 pM, 200 pM, or 100 pM, although higher _KD values may be tolerated if _{the koff} is sufficiently low or the _{kon is} sufficiently high.

In vitro binding affinity assay

Exemplary assays for determining the binding affinity of anti-GPC3 Adnectins include, but are not limited to, solution phase methods such as kinetic exclusion assay (KinExA) (Blake et al., JBC 1996; 271: 27677-85; Drake et al., Anal Biochem 2004; 328: 35-43), surface plasmon resonance (SPR) using a Biacore system (Uppsala, Sweden) (Welford et al., Opt. Quant. Elect 1991; 23: 1; Morton and Myszka, Methods in Enzymology 1998; 295: 268), and homogeneous time-resolved fluorescence (HTRF) assays (Newton et al., J Biomol Screen 2008; 13: 674-82; Patel et al., Assay Drug Dev Technol 2008; 6: 55-68).

In certain embodiments, the interaction of biomolecules can be monitored in real time in a Biacore system, which utilizes SPR to detect the change in resonance angle of light on the surface of a thin gold film on a glass support due to a change in the refractive index up to 300 nm from the surface. Biacore analysis generates association rate constants, dissociation rate constants, equilibrium dissociation constants, and affinity constants. Binding affinity is obtained by evaluating the association rate constant and dissociation rate constant using a Biacore surface plasmon resonance system (Biacore, Inc.). The biosensor chip is activated due to the covalent coupling of the target. The target is then diluted and injected onto the chip to obtain a signal represented by the reaction unit of the fixed material. Since the signal represented by the resonance unit (RU) is proportional to the mass of the fixed material, this represents the range of the fixed target density on the matrix. The binding data and the dissociation data are fitted simultaneously in a global analysis to resolve the net rate expression of a 1:1 bimolecular interaction, thereby generating the best fit values of _kon , koff _{, and Rmax} (maximum response at saturation). The equilibrium dissociation constant, _KD , for binding was calculated from the SPR measurements and expressed as _koff / _kon .

In some embodiments, the anti-GPC3 Adnectins described herein exhibit a KD of 1 μM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 150 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 15 nM or less, 10 nM or less, 5 nM or less, or 1 nM or less in the SPR affinity _assay described in Example 2.

In some embodiments, the anti-GPC3 Adnectin does not substantially bind to closely related proteins, for example, the anti-GPC3 Adnectin does not substantially bind to Glypican 1, Glypican 2, Glypican 4, or Glypican 6.

It should be understood that the assays described above are exemplary and that any method known in the art for determining binding affinity between proteins (e.g., fluorescence resonance energy transfer (FRET), enzyme-linked immunosorbent assay, and competitive binding assay (e.g., radioimmunoassay)) can be used to assess the binding affinity between the anti-GPC3 Adnectins described herein.

Binding Cell Assay

In some embodiments, anti-GPC3 Adnectins and conjugates thereof are internalized into cells expressing Glypican 3. Standard assays for evaluating polypeptide internalization are known in the art and include, for example, the HumZap internalization assay. To assess binding to tumor cells such as Hep-3b or Hep-G2 (ATCC Accession Nos. HB-8064 and HB-8065, respectively), cells can be obtained from publicly available sources such as the American Type Culture Collection and used in standard assays such as flow cytometry analysis.

VII. Drug Conjugates

Also provided are polypeptides comprising an FBS domain conjugated to a therapeutic agent or drug moiety, such as an Adnectin. In an Adnectin-drug conjugate (AdxDC), an FBS moiety (e.g., an anti-GPC3 Adnectin) is conjugated to a drug moiety, wherein the Adnectin acts as a targeting agent to direct the AdxDC to a target cell expressing GPC3, such as a cancer cell. Once the drug is released inside or near the target cell, the drug acts as a therapeutic agent. For a review of the mechanism of action and uses of drug conjugates used with antibodies, such as in cancer treatment, see Schrama et al., Nature Rev. Drug Disc., 5: 147 (2006).

Suitable drug moieties for drug conjugates include cytotoxins or radiotoxins. Cytotoxins or cytotoxic agents include any agent that is harmful to cells (e.g., killing), including antimetabolites, alkylating agents, DNA minor groove binders, DNA chimeras, DNA cross-linking agents, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and antimitotic agents.

Suitable agents include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracenedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin and analogs or homologs thereof. Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil dacarbazine), alkylating agents (e.g., nitrogen mustard, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin D), bleomycin, mithramycin and anthramycin (AMC)) and antimitotic agents (e.g., vincristine and vinblastine). Other preferred examples of therapeutic cytotoxins that can be conjugated to the anti-GPC3 Adnectins of the invention include duocarmycins, calicheamicins, maytansines and auristatins and their derivatives.

Adnectin drug conjugates can be used to modify a given biological response, and the drug moiety is not to be construed as being limited to classical chemotherapeutic agents. For example, the drug moiety can be a protein or polypeptide having a desired biological activity. Such proteins can include, for example, enzymatically active toxins or active fragments thereof, such as abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin; proteins such as tumor necrosis factor or interferon-γ; or biological response modifiers, such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors.

Anti-GPC3 Adnectins can be conjugated to therapeutic agents using linker technology available in the art. Examples of linker types that have been used to conjugate cytotoxins to Adnectins include, but are not limited to, hydrazones, thioethers, esters, disulfides, and peptide-containing linkers. Linkers can be selected that are, for example, sensitive to cleavage at low pH within lysosomal compartments or sensitive to cleavage by proteases, such as proteases preferentially expressed in tumor tissues, such as cathepsins (e.g., cathepsins B, C, D). Examples of cytotoxins are described, e.g., in U.S. Pat. Nos. 6,989,452, 7,087,600, and 7,129,261 and PCT Application Nos. PCT/US02/17210, PCT/US2005/017804, PCT/US06/37793, PCT/US06/060050, PCT/US2006/060711, WO/2006/110476, and U.S. Patent Application No. 60/891,028, all of which are incorporated herein by reference in their entirety.

In certain embodiments, the anti-GPC3 Adnecin and the therapeutic agent are preferably conjugated via a cleavable linker, such as a peptidyl, disulfide bond, or hydrazone linker. More preferably, the linker is a peptidyl linker comprising Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val (SEQ ID NO: 467), Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, or Glu. FBS-DCs may be prepared according to similar methods described in U.S. Pat. Nos. 7,087,600; 6,989,452; and 7,129,261; PCT Publication Nos. WO 02/096910; WO 07/038658; WO 07/051081; WO07/059404; WO 08/083312; and WO 08/103693; U.S. Patent Publication Nos. 2006/0024317; 2006/0004081; and 2006/0247295; the disclosures of which are incorporated herein by reference.

For example, the linker itself can be linked (e.g., covalently linked) to the cysteines of the PmXn module on the anti-GPC3 Adnectin using maleimide chemistry, wherein at least one X is a cysteine. For example, the linker can be covalently linked to the anti-GPC3 Adnectin-PmXn, wherein at least one X is a cysteine. For example, the linker can be linked to the anti-GPC3 Adnectin--PmCn, wherein P is proline, C is cysteine, and m and n are integers of at least 1, such as 1-3. Linkage to cysteine can be performed using maleimide chemistry as known in the art (e.g., Imperiali, B. et al., Protein Engineering: Nucleic Acids and Molecular Biology, Vol. 22, pp. 65-96, Gross, H.J., ed. (2009)). To attach a linker to a cysteine on an anti-GPC3 FBS, the linker may, for example, comprise a maleimido moiety, which then reacts with the cysteine to form a covalent bond. In certain embodiments, the amino acids surrounding the cysteine are optimized to facilitate the chemical reaction. For example, surrounding the cysteine with negatively charged amino acids may result in a faster reaction than surrounding the cysteine with a stretch of positively charged amino acids (EP 1074563). The attachment of the drug moiety to the cysteine on the anti-GPC3 Adnectin is site-specific.

With regard to cancer treatment, the drug is preferably a cytotoxic drug that causes death of targeted cancer cells. Cytotoxic drugs that can be used in anti-GPC3 FBS-DCs include, for example, the following types of compounds and their analogs and derivatives:

a) enediynes, such as calicheamicin (see, e.g., Lee et al., J. Am. Chem. Soc. 1987, 109, 3464 and 3466) and uncialamycin (see, e.g., Davies et al., WO 2007/038868 A2 (2007); Chowdari et al., US 8,709,431 B2 (2012); and Nicolaou et al., WO 2015/023879 A1 (2015));

b) tubulysins (see, e.g., Domling et al., US 7,778,814 B2 (2010); Cheng et al., US 8,394,922 B2 (2013); and Cong et al., US 8,980,824 B2 (2015));

c) DNA alkylating agents, such as analogs of CC-1065 and duocarmycin (see, e.g., Boger, US 6,5458,530 B1 (2003); Sufi et al., US 8,461,117 B2 (2013); and Zhang et al., US 8,852,599 B2 (2014));

d) Epothilones (see, e.g., Vite et al., US 2007/0275904 A1 (2007) and US RE42930E (2011));

e) Auristatins (see, e.g., Senter et al., US 6,844,869 B2 (2005) and Doronina et al., US 7,498,298 B2 (2009));

f) pyrrolobenzodiazepine (PBD) dimers (see, e.g., Howard et al., US 2013/0059800 A1 (2013); US 2013/0028919 A1 (2013); and WO 2013/041606 A1 (2013)); and

g) Maytansinoids, such as DM1 and DM4 (see, for example, Chari et al., US 5,208,020 (1993) and Amphlett et al., US 7,374,762 B2 (2008)).

In addition to disclosing the drug moieties themselves, the aforementioned drug moieties references also disclose linkers suitable for conjugating them that can be used to make drug-linker compounds. Particularly relevant disclosures related to the preparation of drug-linker compounds can be found in Chowdari et al., US 8,709,431 B2 (2012); Cheng et al., US 8,394,922 B2 (2013); Cong et al., US 8,980,824 B2 (2015); Sufi et al., US 8,461,117 B2 (2013); and Zhang et al., US 8,852,599.

Preferably, the drug moiety is a DNA alkylating agent, tubulysin, auristatin, pyrrolobenzodiazepine, enediyne or maytansinoid compound, such as:

In the case of the first five drugs mentioned above, the functional group that enables conjugation is an amine (—NH ₂ ) group, and in the case of the last two drugs, the functional group is a methylamine (—NHMe) group.

In order to conjugate a drug to an adnectin, a linker group is required. The drug is combined with the linker to form a drug-linker compound, which is then conjugated to the adnectin. The drug-linker compound can be represented by formula (I):

in

D is for medicine;

T is a self-immolative group;

t is 0 or 1;

AA ^a and AA ^b are each independently selected from the group consisting of alanine, β-alanine, γ-aminobutyric acid, arginine, asparagine, aspartic acid, γ-carboxyglutamate, citrulline, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, norleucine, norvaline, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine;

p is 1, 2, 3, or 4;

q is 2, 3, 4, 5, 6, 7, 8, 9, or 10;

r is 1, 2, 3, 4, or 5; and

s is 0 or 1.

In Formula II, ^-AAa- [ ^AAb ] _p- represents a polypeptide whose length is determined by the value of p (a dipeptide if p is 1, a tetrapeptide if p is 3, etc.). ^AAa is located at the carboxyl terminus of the polypeptide, and its carboxyl group forms a peptide (amide) bond with the amine nitrogen of drug D (or the self-immolative group T, if present). Conversely, the last AAb is located at the amino terminus of the polypeptide, and its α ^- amino group forms a peptide (amide) bond with the amine nitrogen of drug D (or the self-immolative group T, if present).

Forming peptide bonds,

This depends on whether s is 1 or 0, respectively. Preferred polypeptides ^-AAa- [ ^AAb ] _p- are Val-Cit, Val-Lys, Lys-Val-Ala, Asp-Val-Ala, Val-Ala, Lys-Val-Cit, Ala-Val-Cit, Val-Gly, Val-Gln and Asp-Val-Cit, written in the conventional N to C direction, as _H2N -Val-Cit- _CO2H ). More preferably, the polypeptide is Val-Cit, Val-Lys or Val-Ala. Preferably, the polypeptide ^-AAa- [ ^AAb ] _p- is cleavable by an enzyme found in the target (cancer) cell, such as a cathepsin, in particular cathepsin B.

If the subscript s is 1, the drug-linker (I) contains a poly(ethylene glycol) (PEG) group, which can advantageously improve the solubility of the drug-linker (I) and facilitate conjugation with the adnectin, a step that is performed in aqueous medium. Furthermore, the PEG group can be used as a spacer between the adnectin and the peptide-AA ^a -[AA ^b ] _p -, so that the bulk of the adnectin does not sterically interfere with the action of the peptide cleavage enzyme.

As indicated by the subscript t being equal to 0 or 1, the presence of a self-immolative group T is optional. A self-immolative group is a group such that cleavage of AA ^a or AA ^b (as the case may be) initiates a reaction sequence resulting in the self-immolative group itself being detached from the drug D and releasing the latter to exert its therapeutic function. When present, the self-immolative group T is preferably a p-aminobenzyloxycarbonyl (PABC) group, the structure of which is shown below, wherein the asterisk (*) indicates the end of the PABC bonded to the amine nitrogen of the drug D, and the wavy line It indicates the end bonded to the polypeptide -AA ^a -[AA ^b ] _p -.

Another self-immolative group that can be used is a substituted thiazole as disclosed by Feng in US 7,375,078 B2 (2008).

The maleimide group in formula (I) serves as a reactive functional group for attachment to the adnectin via Michael addition reaction from the thiol group on the adnectin as shown below:

Alternatively, the ε-amino group in the side chain of the lysine residue of the adnectin can be reacted with 2-iminothiolane to introduce a free thiol group (-SH). The thiol group can react with the maleimide group in the drug-linker (I) to achieve conjugation:

Conjugation by this latter method is sometimes referred to as "random conjugation" because the number and position of lysine residues modified by the iminothiolane is difficult to predict.

In certain embodiments, the therapeutic agent in the FBS drug conjugate, such as AdxDC, is tubulysin or tubulysin analogs. Tubulysin belongs to a group of naturally occurring anti-mitotic polypeptides and depsipeptides, including phomopsin, dolastatin and sphaerocephalin (Hamel et al., Curr. Med. Chem.-Anti-Cancer Agents, 2002, 2: 19-53). Tubulysin prevents tubulin from being assembled into microtubules, thereby causing affected cells to be retained in the G2/M phase and undergo apoptosis (Khalil et al., ChemBioChem 2006, 7: 678-683).

In addition to naturally occurring tubulysins, synthetic tubulysin analogs with potent cytotoxic activity suitable for use in the FBS scaffold drug conjugates (eg, AdxDC) provided herein have been described, for example, in US Pat. Nos. 8,394,922 and 8,980,824 (incorporated herein by reference).

In some embodiments, the therapeutic agent in AdxDC is a synthetic tubulysin analog and has a structure represented by Formula (II):

To conjugate a drug moiety (e.g., having Formula II) to a FBS scaffold (e.g., an anti-GPC3 FBS scaffold), a linker moiety having a structure shown in Formula (III) is used:

This linker module comprises a valine-citrulline (Val-Cit, written in the conventional N to C orientation) dipeptide that is designed to be cleaved by the intracellular enzyme cathepsin B after AdxDC reaches the target cancer cells and is internalized, thereby releasing the therapeutic agent to exert its cytotoxic effect (Dubowchik et al., Biorg. Med. Chem Lett., 1998, 8:3341-3346; Dubowchik et al., Biorg. Med. Chem Lett., 1998, 8:3347-3352; Dubowchik et al., Bioconjugate Chem., 2002, 13:855-869).

The drug (II) and the linker (III) are coupled to produce a drug-linker compound having a structure represented by formula (IV), which is then conjugated to the adnectin. The preparation of drug-linkers (IV) is described in US 8,394,922 B2 (2013) by Cheng et al. (see, e.g., FIG. 20b and Example 22), the disclosure of which is incorporated herein. One skilled in the art will appreciate that in the case of drug-linkers (IV), there are no optional self-immolative groups or PEG groups, but these groups may be incorporated if desired.

In the preparation of the AdxDC conjugate, a therapeutic agent-linker compound having a structure represented by Formula (IV) was prepared and then conjugated to the FBS scaffold.

In some embodiments, the FBS-drug conjugate has a structure represented by Formula (V).

wherein m is 1, 2, 3, 4 or greater. In certain embodiments, m is 1. In other embodiments, m is 2.

In one embodiment, the sulfhydryl group in the side chain of the C-terminal cysteine residue of the anti-GPC3 Adnectin (Adx) reacts with the maleimide group in compound (V) to achieve conjugation:

In another embodiment, the thiol groups of the two cysteines located at the C-terminus of the anti-GPC3 Adnectin (Adx) react with the maleimide group in compound (IV) to achieve conjugation of two drug molecules per Adnectin (eg DAR2, FIG. 2 ).

In some embodiments, the anti-GPC3 Adx in the conjugate has an amino acid sequence as described above in Section IA, which has been modified to contain a C-terminal tail comprising cysteine. In some embodiments, the anti-GPC3 Adx comprises a core amino acid sequence selected from SEQ ID NO: 5, 9-10, 18, 22-23, 31, 35-36, 44, 48-49, 57, 61-62, 70, 74-75, 83, 87-88, 98, 102-105, 128, 130-132, 155, 157-159, 180, 184-186, 20-, 211-213, 236, 238-240, 263, 265-267, 290, 292-294, 317, and 319-321, which has been modified to contain a C-terminal module comprising cysteine.

In some _embodiments , the anti-GPC3Adx has been modified to contain a C-terminal module consisting of _PmCXn or _PmCXn1CXn2 as defined herein. In certain embodiments, the C-terminal module consists of PC, PCC, or any one of the C-terminal sequences described herein _, such as the amino acid sequences listed in SEQ ID NO: 409-423. In certain embodiments, _the C-terminal module consists of PC or PCPPPPPC (SEQ ID NO: 416).

In certain embodiments, the modified anti-GPC3 Adx comprises a C-terminal module consisting of _PmCXn and is selected from SEQ ID NOs: 11-14, 24-27, _37-40 , 50-53, 63-66, 76-79, 89-93, 106-118, 133-145, 160-172, 187-199, 214-226, 241-253, 268-280, 295-307, and 322-334.

In certain embodiments, the modified anti-GPC3 Adx comprises a C-terminal module consisting of _PmCXn1CXn2 and is selected from SEQ ID NOs: 15-17 _, 28-30, 41-43, 54-57, 67-70, 80-83, 94-97, 119-127, 146-154, 173-181, 200-208, 227-235, 254-262, _281-289 , 308-316, and 335-343.

In specific embodiments, the anti-GPC3 Adx used in the drug conjugate is any one of SEQ ID NOs: 114-118; 123-127; 141-145; 150-154; 168-172; 177-181; 195-199; 204-208; 222-226; 231-235; 249-253; 258-262; 276-280; 285-289; 303-307; 312-316; 330-334; and 339-343.

The anti-GPC3 Adnectins described herein can also be conjugated to radioisotopes to produce cytotoxic radiopharmaceuticals. Examples of radioisotopes that can be conjugated to antibodies for diagnosis or treatment include, but are not limited to, iodine ¹³¹ , indium ¹¹¹ , yttrium ⁹⁰ , and lutetium ^177. Methods for preparing radioconjugates are established in the art.

VIII. Pharmaceutical Compositions

Also provided are pharmaceutically acceptable compositions comprising the anti-GPC3 Adnectins and conjugates described herein, wherein the compositions are substantially endotoxin-free and/or pyrogen-free.

Methods for preparing formulations well known in the art are found in, for example, "Remington: The Science and Practice of Pharmacy" (20th ed., ed. A. R. Gennaro A R., 2000, Lippincott Williams & Wilkins, Philadelphia, Pa.). Formulations for parenteral administration may, for example, contain excipients, sterile water, saline, polyalkylene glycols (such as polyethylene glycol), oils of plant origin, or hydrogenated naphthalene. Biocompatibility, biodegradable lactide polymers, lactide/glycolide copolymers, or polyoxyethylene-polyoxypropylene copolymers can be used to control the release of the compound. Nanoparticle formulations (e.g., biodegradable nanoparticles, solid lipid nanoparticles, liposomes) can be used to control the biodistribution of the compound. Other potentially useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. The concentration of the compound in the formulation varies depending on many factors, including the dose and route of administration of the drug to be administered.

Therapeutic formulations containing the protein are prepared for storage by mixing the described protein of the desired purity with optional physiologically acceptable carriers, excipients or stabilizers (Osol, A., ed., Remington's Pharmaceutical Sciences, 16th edition (1980)), in the form of aqueous solutions, lyophilized or other dry preparations. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextran; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants such as Tween, or polyethylene glycol (PEG).

The polypeptide may optionally be administered in the form of a pharmaceutically acceptable salt, such as a non-toxic acid addition salt or metal complex commonly used in the pharmaceutical industry. Examples of acid addition salts include organic acids such as acetic acid, lactic acid, pamoic acid, maleic acid, citric acid, malic acid, ascorbic acid, succinic acid, benzoic acid, palmitic acid, suberic acid, salicylic acid, tartaric acid, methanesulfonic acid, toluenesulfonic acid, or trifluoroacetic acid, etc.; polymeric acids such as tannic acid, carboxymethyl cellulose, etc.; and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, etc. Metal complexes include zinc, iron, etc. In one example, the polypeptide is formulated in the presence of sodium acetate to increase thermal stability.

The formulations herein may also contain more than one active compound when required for the particular indication being treated, preferably, the active compounds have complementary activities that do not adversely affect each other. Such molecules are suitably present in combination in amounts that are effective for the intended purpose.

Proteins can also be embedded in prepared microcapsules, such as hydroxymethylcellulose or gelatin microcapsules and polymethylmethacrylate microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions, for example, by coacervation techniques or by interfacial polymerization. Such techniques are disclosed in Osol, A., ed., Remington's Pharmaceutical Sciences, 16th edition (1980).

Preparations for in vivo administration must be sterile. This is easily achieved by sterile filtration membranes.

Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the proteins described herein, in the form of shaped articles, such as films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and L-glutamic acid-γ-ethyl ester, non-degradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. Although polymers such as ethylene vinyl acetate and lactic acid-glycolic acid enable sustained release of molecules for more than 100 days, certain hydrogels release proteins for shorter periods of time. When encapsulated proteins are retained in the body for a long time, they may denature or aggregate due to exposure to moisture at 37°C, resulting in loss of biological activity and possible changes in immunogenicity. Rational strategies can be designed for stabilization based on the mechanisms involved. For example, if the aggregation mechanism is found to be intermolecular S--S bond formation through thiol-disulfide bond conversion, stabilization can be achieved by modifying thiol residues, lyophilizing acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

For therapeutic use, the protein is administered to a subject in a pharmaceutically acceptable dosage form. The protein can be administered intramuscularly, subcutaneously, intraarticularly, intrasynovially, intrathecally, orally, topically, or by inhalation, either by bolus or by continuous intravenous administration over a period of time. The protein can also be administered intratumorally, peritumorally, intralesionally, or peritumorally to exert local as well as systemic therapeutic effects. Suitable pharmaceutically acceptable carriers, diluents, and excipients are well known and can be determined by one skilled in the art when the clinical situation permits. Suitable carriers, diluents, and/or excipients include: (1) Dulbecco's phosphate buffered saline, pH about 7.4, containing 1 mg/ml to 25 mg/ml human serum albumin, (2) 0.9% saline (0.9% w/v NaCl), and (3) 5% (w/v) dextrose. The methods of the present invention can be practiced in vitro, in vivo, or ex vivo.

The administration of the protein, and one or more additional therapeutic agents, whether co-administered or sequentially, can be carried out as described above for therapeutic applications. The skilled artisan will appreciate that suitable pharmaceutically acceptable carriers, diluents, and excipients for co-administration depend on the identity of the specific therapeutic agents being co-administered.

When present in aqueous dosage forms other than lyophilized dosage forms, the protein is generally formulated to a concentration of about 0.1 mg/ml to 100 mg/ml, although wide variations outside these ranges are permitted. For treatment of disease, the appropriate protein dosage will depend on the type of disease to be treated, the severity and course of the disease, whether the protein is administered for preventive or therapeutic purposes, the course of previous treatments, the patient's clinical history and response to the fusion, and the judgment of the attending physician. The protein is suitably administered to the patient at one time or over a series of treatments.

A therapeutically effective dose refers to a dose that produces the therapeutic effect for which the administration is intended. The exact dose will depend on the condition to be treated and can be determined by a person skilled in the art using known techniques. A preferred dose range may be from about 10 mg/m2 to about 2000 mg/m2, more preferably from about 50 mg/m2 to about 1000 mg/m2. In some embodiments, an anti-GPC3 Adnectin or anti-GPC3 AdxDC is administered at about 0.01 μg/kg to about 50 mg/kg per day, 0.01 mg/kg to about 30 mg/kg per day, or 0.1 mg/kg to about 20 mg/kg per day.

Anti-GPC3 Adnectin or anti-GPC3 AdxDC can be administered daily (e.g., once, twice, three times, or four times a day) or less frequently (e.g., once every other day, once or twice a week, once every two weeks, once every three weeks, or once a month). In addition, as is known in the art, adjustments may need to be made for age and weight, general health, sex, diet, dosing time, drug interactions, and disease severity, and can be determined by those skilled in the art using routine experiments.

IX. Treatment Methods

The anti-GPC3 Adnectins and drug conjugates thereof described herein are useful for treating cancers having tumor cells expressing GPC3 (e.g., high levels of GPC3 relative to healthy tissue). In some embodiments, the cancer is selected from liver cancer (e.g., hepatocellular carcinoma (HCC) or hepatoblastoma), melanoma, sarcoma, lung cancer (e.g., squamous lung cancer), and Wilms' tumor. In addition, the GPC3 Adnectins described herein are useful for treating refractory or recurrent malignancies.

As used herein, the term "subject" is intended to include human or non-human animals. Preferred subjects include human patients with a disorder mediated by GPC3 activity or undesirable cells expressing high levels of GPC3. When an anti-GPC3 Adnectin or drug conjugate thereof is administered with another agent, the two may be administered in either order or simultaneously.

For example, anti-GPC3 Adnectins, multispecific or bispecific molecules and drug conjugates thereof can be used to elicit one or more of the following biological activities in vivo or in vitro: inhibiting the growth of and/or killing cells expressing GPC3; mediating phagocytosis or ADCC of cells expressing GPC3 in the presence of human effector cells, or possibly modulating GPC3 activity, for example, by blocking binding of a GPC3 ligand to GPC3.

In certain embodiments, the anti-GPC3 Adnectins or anti-GPC3 AdxDCs described herein are combined with immunogenic agents such as cancer cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), antigen presenting cells such as dendritic cells carrying tumor-associated antigens, and preparations of cells transfected with genes encoding immunostimulatory cytokines (He et al., 2004). Non-limiting examples of tumor vaccines that can be used include peptides of melanoma antigens such as gp100, MAGE antigens, Trp-2, MARTI, and/or tyrosinase, or transfected tumor cells expressing the cytokine GM-CSF. GPC3 blockade can also be combined with standard cancer treatments (including chemotherapeutic regimens, radiation therapy, surgery, hormone deprivation, and angiogenesis inhibitors) as well as another immunotherapeutic agent (e.g., anti-PD-1, anti-CTLA-4, and anti-LAG-3 Adnectins or antibodies).

Provided herein are combination treatment methods wherein an anti-GPC3 Adnectin and/or anti-GPC3 AdxDC is administered (simultaneously or sequentially) with one or more additional agents (e.g., small molecule drugs, antibodies, or antigen-binding portions thereof) effective in stimulating an immune response, thereby enhancing, stimulating, or upregulating an immune response in a subject.

Typically, for example, anti-GPC3 Adnectin or/or anti-GPC3 AdxDC described herein can be combined with a tumor immunotherapy agent, such as an agonist of (i) a stimulatory (e.g., co-stimulatory) molecule (e.g., a receptor or ligand) on an immune cell such as a T cell and/or an antagonist of (ii) an inhibitory signal or molecule (e.g., a receptor or ligand), both of which result in an immune response, such as an amplification of an antigen-specific T cell response. In certain aspects, the tumor immunotherapy agent is an agonist of (i) a stimulatory (including co-stimulatory) molecule (e.g., a receptor or ligand) or an antagonist of (ii) an inhibitory (including co-inhibitory) molecule (e.g., a receptor or ligand) on a cell involved in innate immunity, such as a NK cell, and wherein the tumor immunotherapy agent enhances innate immunity. Such tumor immunotherapy agents are generally referred to as immune checkpoint regulators, such as immune checkpoint inhibitors or immune checkpoint stimulators.

In certain embodiments, anti-GPC3 Adnectins and/or anti-GPC3 AdxDCs are administered with agents targeting stimulatory or inhibitory molecules that are members of the immunoglobulin superfamily (IgSF). For example, anti-GPC3 Adnectins and/or anti-GPC3 AdxDCs, such as those described herein, can be administered to a subject together with agents targeting IgSF family members to increase an immune response. For example, anti-GPC3 Adnectins and/or anti-GPC3 AdxDCs can be administered together with agents targeting (or specifically binding to) members of the membrane-bound ligand B7 family, including B7-1, B7-2, B7 (PD-L1), B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6 or co-stimulatory or co-inhibitory receptors that specifically bind to B7 family members.

Anti-GPC3 Adnectin and/or anti-GPC3 AdxDC can also be administered with agents that target TNF and TNFR family members of molecules (ligands or receptors), such as CD40 and CD40L, OX-40, GITR, GITRL, OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137, TRAIL/Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEA KR/Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTβR, LIGHT, DcR3, HVEM, VEGI/TL1A, TRAMP/DR3, EDA1, EDA2, TNFR1, lymphotoxin α/TNFβ, TNFR2, TNFα, LTβR, lymphotoxin α1β2, FAS, FASL, RELT, DR6, TROY, and NGFR (see, e.g., Tansey (2009) Drug Discovery Today 00:1).

T cell responses can be stimulated by administering one or more of the following agents:

(1) antagonists (inhibitors or blockers) of proteins that inhibit T cell activation (e.g., immune checkpoint inhibitors), such as CTLA-4, PD-1, PD-L1, PD-L2, and LAG-3 as described above, and any of the following proteins: TIM-3, galectin-9, CEACAM-1, BTLA, CD69, galectin-1, TIGIT, CD113, GPR56, VISTA, B7-H3, B7-H4, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, and TIM-4; and/or

(2) Agonists of proteins that stimulate T cell activation, such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, DR3, and CD28H.

Exemplary agents that modulate one of the above proteins and can be combined with anti-GPC3 Adnectins and/or anti-GPC3 AdxDCs (e.g., those described herein for treating cancer) include: Yervoy ^™ (ipilimumab) or tremelimumab (targeting CTLA-4), galiximab (targeting B7.1), BMS-936558 (targeting PD-1), MK-3475 (targeting PD-1), AMP224 (targeting B7DC), BMS-936559 (targeting B7-H1), MPDL3280A (targeting B7-H1), MEDI-570 (targeting ICOS), AMG557 (targeting B7H2), MGA271 (targeting B7H3), IMP321 (targeting LAG-3), BMS-663513 (targeting CD137), PF-05082566 (targeting CD137), CDX-1127 (targeting CD27), anti-OX40 antibody (Providence HealthServices), huMAbOX40L (targeting OX40L), atacicept (targeting TACI), CP-870893 (targeting CD40), rucamumab (targeting CD40), daclizumab (targeting CD40), muromonab-CD3 (targeting CD3), ipilimumab (targeting CTLA-4), and/or MK4166.

Anti-GPC3 Adnectin or anti-GPC3 AdxDC can also be administered with pidilizumab (CT-011), although its specificity for PD-1 binding has been questioned.

Other molecules that can be combined with anti-GPC3 Adnectin or anti-GPC3 AdxDC for the treatment of cancer include antagonists of inhibitory receptors on NK cells or agonists of activating receptors on NK cells. For example, an anti-GITR agonist antibody can be combined with an antagonist of KIR (e.g., rilulumab).

T cell activation is also regulated by soluble cytokines, and anti-GPC3 Adnectin or anti-GPC3 AdxDC can be administered to a subject, for example, with cancer, together with an antagonist of a cytokine that inhibits T cell activation or an agonist of a cytokine that stimulates T cell activation.

In certain embodiments, an anti-GPC3 Adnectin or anti-GPC3 AdxDC can be combined with: (i) an antagonist (or inhibitor or blocker) of a protein of the IgSF family or the B7 family or the TNF family that inhibits T cell activation, or an antagonist of a cytokine that inhibits T cell activation (e.g., IL-6, IL-10, TGF-β, VEGF; "immunosuppressive cytokines") and/or (ii) an agonist of a stimulatory receptor of the IgSF family, the B7 family or TNF, or an agonist of a cytokine that stimulates T cell activation, for stimulating an immune response, e.g., for treating a proliferative disease such as cancer.

Other agents for combination therapy include agents that inhibit or deplete macrophages or monocytes, including but not limited to CSF-1R antagonists, such as CSF-1R antagonist antibodies, including RG7155 (WO11/70024, WO11/107553, WO11/131407, WO13/87699, WO13/119716, WO13/132044) or FPA-008 (WO11/140249; WO13169264; WO14/036357).

Anti-GPC3 Adnectin or anti-GPC3 AdxDC can also be administered with an agent that inhibits TGF-β signaling.

Additional agents that can be combined with anti-GPC3 Adnectins and/or anti-GPC3 AdxDCs include agents that enhance tumor antigen presentation, such as dendritic cell vaccines, GM-CSF-secreting cellular vaccines, CpG oligonucleotides, and imiquimod, or treatments that enhance the immunogenicity of tumor cells (e.g., anthracyclines).

Other treatments that may be combined with anti-GPC3 Adnectins or anti-GPC3 AdxDCs include treatments that deplete or block Treg cells, such as agents that specifically bind to CD25.

Another treatment that can be combined with an anti-GPC3 Adnectin or anti-GPC3 AdxDC is one that inhibits metabolic enzymes such as indoleamine dioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthase.

Another class of agents that may be used with anti-GPC3 Adnectins or/and anti-GPC3 AdxDCs include agents that inhibit adenosine formation or inhibit adenosine A2A receptors.

Other therapies that may be combined with anti-GPC3 Adnectins or anti-GPC3 AdxDCs for treating cancer include therapies that reverse/prevent T cell anergy or exhaustion and therapies that trigger innate immune activation and/or inflammation at tumor sites.

Anti-GPC3 Adnectin or anti-GPC3 AdxDC can be combined with more than one tumor immunotherapy agent, and can be combined, for example, with a combination approach targeting multiple components of immune pathways, such as one or more of the following: treatments that enhance tumor antigen presentation (e.g., dendritic cell vaccines, cellular vaccines that secrete GM-CSF, CpG oligonucleotides, imiquimod); treatments that inhibit negative immune regulation, such as by inhibiting CTLA-4 and/or PD1/PD-L1/PD-L2 pathways and/or depleting or blocking Treg or other immunosuppressive cells; treatments that stimulate positive immune regulation, such as with agonists that stimulate CD-137, OX-40 and/or GITR pathways and/or stimulate T cell effector function; treatments that systemically increase the frequency of anti-tumor T cells; depleting Therapies that deplete or inhibit Tregs (such as Tregs in tumors), for example, using CD25 antagonists (e.g., daclizumab) or by ex vivo anti-CD25 bead depletion; therapies that affect suppressive myeloid cell function in tumors; therapies that enhance the immunogenicity of tumor cells (e.g., anthracyclines); adoptive T cell or NK cell transfer, including genetically modified cells, such as cells modified with chimeric antigen receptors (CAR-T therapy); therapies that inhibit metabolic enzymes such as indoleamine dioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthase; therapies that reverse/prevent T cell anergy or exhaustion; therapies that trigger innate immune activation and/or inflammation at the tumor site; administration of immunostimulatory cytokines; or blockade of immunosuppressive cytokines.

The anti-GPC3 Adnectins and/or anti-GPC3 AdxDCs described herein can be used with one or more of the following agents: agonists that bind to positive co-stimulatory receptors; blockers, antagonists, and one or more agents that systemically increase the frequency of anti-tumor T cells by attenuating signal transduction through inhibitory receptors; agents that overcome different immunosuppressive pathways in the tumor microenvironment (e.g., blocking inhibitory receptor engagement (e.g., PD-L1/PD-1 interaction)); depletion or inhibition of Tregs (e.g., using anti-CD25 monoclonal antibodies (e.g., daclizumab) or by ex vivo anti-CD25 bead depletion), inhibition of metabolic enzymes such as IDO; or reversal/prevention of T cell anergy or exhaustion) and agents that trigger innate immune activation and/or inflammation at the tumor site.

Provided herein is a use of any anti-GPC3 Adnectin described herein in the preparation of a medicament for treating a subject having cancer. The present disclosure provides a medical use of any anti-GPC3 Adnectin described herein corresponding to all embodiments of the methods of treatment employing the anti-GPC3 Adnectin described herein.

XI. Detectable Labels

The anti-GPC3 Adnectins described herein can also be used in various diagnostic and imaging applications. In certain embodiments, the anti-GPC3 Adnectin is labeled with an in vivo detectable moiety, and the Adnectin so labeled can be used as an in vivo imaging agent, for example, for whole body imaging. For example, in one embodiment, a method for detecting a GPC3-positive tumor in a subject comprises: administering to a subject an anti-GPC3 Adnectin linked to a detectable marker, and detecting the marker in the subject after an appropriate time.

Anti-GPC3 Adnectin imaging agents can be used to diagnose conditions or diseases associated with increased levels of GPC3, such as cancer, where tumors selectively overexpress GPC3. In a similar manner, anti-GPC3 Adnectins can be used to monitor GPC3 levels in a subject (e.g., a subject undergoing treatment to reduce GPC3 levels and/or GPC3-positive cells (e.g., tumor cells). Anti-GPC3 Adnectins can be used with or without further modification and can be labeled by covalent or non-covalent attachment of a detectable moiety.

The detectable label can be any of the various types currently used in the field of in vitro diagnostics, including particulate labels, including metal sols, such as colloidal gold; isotopes, such as I ¹²⁵ or Tc ⁹⁹ , for example provided with peptide chelators of the type N ₂ S ₂ , N ₃ S or N ₄ ; chromophores, including fluorescent markers, biotin, luminescent markers, phosphorescent markers, etc., as well as enzyme labels that convert a given substrate into a detectable marker, and polynucleotide tags that are revealed by subsequent amplification, such as polymerase chain reaction. The biotinylated anti-GPC3 FBS can then be detected by avidin or streptavidin binding. Suitable enzyme labels include horseradish peroxidase, alkaline phosphatase, and the like. For example, the marker can be an enzyme (alkaline phosphatase) which can be detected by converting 1,2-dioxetane substrates such as adamantyl methoxyphosphophenyl dioxetane (AMPPD), 3-(4-(methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro)tricyclo{3.3.1.1 3,7}decane}-4-yl)phenyl phosphate disodium (CSPD), and CDP and Or other luminescent substrates known to those skilled in the art, such as suitable chelates of the lanthanides terbium (III) and europium (III), are detected by the presence or formation of chemiluminescence.

Detectable moieties that can be used include radioactive substances, such as radioactive heavy metals, such as iron chelates, radioactive chelates of gadolinium or manganese, positron emitters of oxygen, nitrogen, iron, carbon or gallium, ¹⁸ F, 60 Cu, ⁶¹ Cu, ⁶² Cu, ⁶⁴ Cu, ¹²⁴ I, ⁸⁶ Y, ⁸⁹ Zr, ⁶⁶ Ga, ⁶⁷ Ga, ⁶⁸ Ga, ⁴⁴ Sc, ⁴⁷ Sc, ¹¹ C, ¹¹¹ In, ^114m In, ¹¹⁴ In, ¹²⁵ I, ¹²⁴ I, ^{131 I, 123 I} , ¹³¹ I, ¹²³ I, ³² Cl, ³³ Cl ^{, 34} Cl, ⁷⁴ Br, ^{75 Br, 76} Br, ⁷⁷ Br, ⁷⁸ Br, ⁸⁹ Zr, ¹⁸⁶ Re, ¹⁸⁸ Re, ⁸⁶ Y, ⁹⁰ Y, ¹⁷⁷ Lu, ⁹⁹ Tc, ²¹² Bi, ²¹³ Bi, ²¹² Pb, ²²⁵ Ac, or ¹⁵³ Sm.

The means of detection is determined by the label chosen. Where the label is particulate and accumulates at appropriate levels, the label or its reaction products may be visualized by the naked eye or by the use of instrumentation such as a spectrophotometer, luminometer, fluorometer, etc., all in accordance with standard practice.

The detectable module can be connected to cysteine according to methods known in the art. When the detectable module is a radioactive material, such as those further described herein, the detectable module can be connected to FBS by a chelating agent that reacts with cysteine, such as a chelating agent containing maleimide, such as maleimide-NODAGA or maleimide-DBCO. Maleimide-NODAGA or maleimide-DBCO can react with the cysteine on the C-terminus of FBS (e.g., by a PmXn module, wherein at least one X is cysteine) to produce FBS-NODAGA or FBS-DBCO, respectively. Any of the following chelators may be used provided that it comprises or can be modified to comprise a reaction module that reacts with cysteine: DFO, DOTA and its derivatives (CB-DO2A, 3p-C-DEPA, TCMC, Oxo-DO3A), TE2A, CB-TE2A, CB-TE1A1P, CB-TE2P, MM-TE2A, DM-TE2A, diamsar and derivatives, NODASA, NODAGA, NOTA, NETA, TACN-TM, DTPA, 1B4M-DTPA, CHX-A"-DTPA, TRAP (PRP9), NOPO, AAZTA and derivatives (DATA), _H2dedpa , _H4octapa , _H2azapa , _{H5decapa, H6phospa ,} HBED, SHBED, BPCA, CP256, PCTA, HEHA, PEPA, EDTA, TETA, and TRITA-based chelators, and close analogs and derivatives thereof.

In certain embodiments, FBS is labeled with a PET tracer and used as an in vivo imaging agent. For example, FBS can be labeled with the PET tracer ⁶⁴ Cu. ⁶⁴ Cu can be attached to FBS with a C-terminal cysteine using a chelating agent such as maleimide-NODAGA.

To synthesize the anti-GPC3 Adnectin-based imaging agents described herein, other methods recognized in the art for labeling polypeptides with radionuclides such as ⁶⁴ Cu and ¹⁸ F may also be used. See, e.g., US2014/0271467; Gill et al., Nature Protocols 2011; 6: 1718-25; Berndt et al., Nuclear Medicine and Biology 2007; 34: 5-15, Inkster et al., Bioorganic & Medicinal Chemistry Letters 2013; 23: 3920-6, the contents of which are incorporated herein by reference in their entirety.

In certain embodiments, the GPC3 imaging agent comprises a PEG molecule (e.g., 5KDa PEG, 6KDa PEG, 7KDa PEG, 8KDa PEG, 9KDa PEG, or 10KDa PEG) to increase the blood PK of the imaging agent in small increments to enhance imaging contrast or to increase the affinity of the anti-GPC3 Adnectin-based imaging agent.

XI. Detection of GPC3

In vivo detection methods

In certain embodiments, GPC3-positive cells or tissues (e.g., tumors expressing GPC3) can be imaged using labeled anti-GPC3 Adnectins. For example, labeled anti-GPC3 Adnectins are administered to a subject in an amount sufficient to allow the labeled Adnectin to be taken up into the tissue of interest (e.g., tumors expressing GPC3). The subject is then imaged using an imaging system such as PET for an amount of time appropriate to the specific radionuclide used. Cells or tissues expressing GPC3, such as tumors expressing GPC3, to which the labeled anti-GPC3 Adnectin binds are then detected by the imaging system.

PET imaging with a GPC3 imaging agent can be used to qualitatively or quantitatively detect GPC3. A GPC3 imaging agent can be used as a biomarker, and the presence or absence of a GPC3 positive signal in a subject can indicate, for example, that the subject will respond to a given treatment (e.g., a cancer treatment), or that the subject is responding or not responding to the treatment.

In certain embodiments, the progression or regression of a disease (e.g., a tumor) over time or as a function of treatment can be imaged. For example, the size of a tumor in a subject undergoing cancer treatment (e.g., chemotherapy, radiotherapy) can be monitored, and based on the detection of a labeled anti-GPC3 Adnectin, the extent of tumor regression can be monitored in real time.

An effective amount of an imaging agent (e.g., ¹⁸ F-Adnectin imaging agent, ⁶⁴ Cu-Adnectin imaging agent) that results in uptake into cells or tissues of interest (e.g., tumors) can depend on a variety of factors, including, for example, the age, weight, general health, sex, and diet of the host; the time of administration; the route of administration; the excretion rate of the specific probe employed; the duration of treatment; the presence of other drugs used in combination or concomitantly with the particular composition employed; and other factors.

In certain embodiments, imaging of tissue expressing GPC3 is achieved before, during, and after administration of a labeled anti-GPC3 Adnectin.

In certain embodiments, the anti-GPC3 Adnectins described herein can be used for PET imaging of the lung, heart, kidney, liver, and skin, among other organs, or tumors associated with these organs that express GPC3.

In certain embodiments, the anti-GPC3 imaging agent provides a contrast of at least 50%, 75%, 2, 3, 4, 5 or more. The examples show that all anti-GPC3 Adnectins used provide a PET contrast of 2 or more and that the affinity of the Adnectin is not important.

When used with short half-life radionuclides (eg, ¹⁸ F) for imaging (eg, PET), the radiolabeled anti-GPC3 Adnectin is preferably administered intravenously. Other routes of administration may also be suitable, depending on the half-life of the radionuclide used.

In certain embodiments, the anti-GPC3 imaging agents disclosed herein are used to detect GPC3-positive cells in a subject by administering an anti-GPC3 imaging agent disclosed herein to the subject and detecting the imaging agent, wherein the detected imaging agent defines the location of the GPC3-positive cells in the subject. In certain embodiments, the imaging agent is detected by positron emission tomography.

In certain embodiments, the anti-GPC3 imaging agents disclosed herein are used to detect a GPC3-expressing tumor in a subject by administering an anti-GPC3 imaging agent disclosed herein to the subject and detecting the imaging agent, wherein the detected imaging agent defines the location of the tumor in the subject. In certain embodiments, the imaging agent is detected by positron emission tomography.

In certain embodiments, images of the anti-GPC3 imaging agents described herein are obtained by administering the imaging agent to a subject and imaging the distribution of the imaging agent in vivo by positron emission tomography.

Thus, provided herein are methods of obtaining quantitative images of tissues or cells expressing GPC3, the methods comprising contacting the cells or tissues with an anti-GPC3 imaging agent described herein, and detecting or quantifying tissues expressing GPC3 using positron emission tomography.

Also provided herein is a method for detecting a tumor expressing GPC3, comprising administering an imaging-effective amount of an anti-GPC3 imaging agent described herein to a subject having a tumor expressing GPC3, and detecting radioactive emission of the imaging agent in the tumor using positron emission tomography, wherein the radioactive emission is detected in the tumor.

Also provided herein are methods of diagnosing the presence of a tumor expressing GPC3 in a subject, the method comprising:

● administering an anti-GPC3 imaging agent described herein to a subject in need thereof; and

● obtaining a radiographic image of at least a portion of the subject to detect the presence or absence of the imaging agent;

- Wherein the presence and location of the imaging agent above the background indicates the presence and location of disease.

Also provided herein is a method of monitoring the progress of an anti-tumor therapy for a GPC3-expressing tumor in a subject, the method comprising:

● administering an anti-GPC3 imaging agent described herein to a subject in need thereof at a first time point and obtaining an image of at least a portion of the subject to determine the size of the tumor;

● administering an anti-tumor therapy to the subject;

- administering the imaging agent to the subject at one or more subsequent time points, and obtaining an image of at least a portion of the subject at each time point;

• Wherein the size and location of the tumor at each time point is indicative of the progression of the disease.

In vitro detection methods

In addition to detecting GPC3 in vivo, anti-PDL1 Adnectins such as those described herein can be used to detect target molecules in a sample. The method may include contacting the sample with an anti-GPC3 Adnectin described herein, wherein the contact is performed under conditions that allow the formation of an anti-GPC3 Adnectin-target complex; and detecting the complex, thereby detecting the target in the sample. Detection can be performed using any art-recognized technique, such as radiography, immunoassay, fluorescence detection, mass spectrometry, or surface plasmon resonance. The sample may be from a human or other mammal. For diagnostic purposes, suitable substances are detectable labels including radioisotopes for whole-body imaging, as well as radioisotopes, enzymes, fluorescent markers and other suitable antibody labels for sample testing.

The detectable label can be any of the various types currently used in the field of in vitro diagnostics, including particulate labels, including metal sols, such as colloidal gold; isotopes, such as I ¹²⁵ or Tc ⁹⁹ , for example provided with peptide chelators of the N ₂ S ₂ , N ₃ S or N ₄ type; chromophores, including fluorescent markers, biotin, luminescent markers, phosphorescent markers, etc., as well as enzyme labels that convert a given substrate into a detectable marker, and polynucleotide tags that are revealed by subsequent amplification, such as polymerase chain reaction. Biotinylated FBS can then be detected by avidin or streptavidin binding. Suitable enzyme labels include horseradish peroxidase, alkaline phosphatase, and the like. For example, the marker can be an enzyme (alkaline phosphatase) which can be detected by converting 1,2-dioxetane substrates such as adamantyl methoxyphosphophenyl dioxetane (AMPPD), 3-(4-(methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo{3.3.1.1 3,7}decane}-4-yl)phenyl phosphate disodium (CSPD), and CDP and Or other luminescent substrates well known to those skilled in the art, such as suitable lanthanide terbium (III) and europium (III) chelates, are detected by the presence or formation of chemiluminescence. Other labels include those listed in the imaging section above. The detection means is determined by the selected label. In the case where the label is particulate and accumulates at an appropriate level, the appearance of the label or its reaction product can be obtained by the naked eye or using instruments such as spectrophotometers, luminometers, fluorometers, etc., all following standard practice.

XII. Kits and Products

The anti-GPC3 Adnectins and drug conjugates thereof described herein may be provided in a kit, which is a packaged combination of predetermined amounts of reagents with instructions for use in the therapeutic or diagnostic methods described herein.

For example, in certain embodiments, an article containing materials for treating or preventing a disorder or condition described herein or for a detection method described herein is provided. The article includes a container and a label. Suitable containers include, for example, bottles, vials, syringes, test tubes, and the like. The container can be formed of various materials such as glass or plastic. The container can hold a composition described herein for in vivo imaging and can have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The active agent in the composition is an anti-GPC3 Adnectin or anti-GPC3 AdxDC described herein. The article can further include a second container containing a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, and dextrose solution. It can further include other items required from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

Exemplary embodiments

1. A polypeptide comprising a fibronectin-based scaffold (FBS) comprising BC, DE and FG loops, wherein one or more of the loops are altered relative to the corresponding loops of a wild-type FBS domain, and wherein the polypeptide specifically binds to human glypican 3 (GPC3) with a _KD of 1 μM or less.

2. The polypeptide of embodiment 1, wherein the FBS is a fibronectin type III (Fn3) domain.

3. The polypeptide of embodiment 2, wherein the Fn3 domain is the tenth ( ¹⁰ Fn3) domain of human fibronectin type III.

4. The polypeptide according to any one of the preceding embodiments, wherein the BC loop comprises an amino acid sequence selected from the group consisting of:

(a) SEQ ID NO: 6, 19, 32, 45, 58, 71, 84 or 99; and

(b) the BC loop has 1, 2 or 3 amino acid substitutions, insertions or deletions relative to the BC loop of SEQ ID NO: 6, 19, 32, 45, 58, 71, 84 or 99.

5. The polypeptide according to any one of the preceding embodiments, wherein the DE loop comprises an amino acid sequence selected from the group consisting of:

(a) SEQ ID NO:7, 20, 33, 46, 59, 72, 85 or 100; and

(b) the DE loop has 1, 2 or 3 amino acid substitutions, insertions or deletions relative to the DE loop of SEQ ID NO: 7, 20, 33, 46, 59, 72, 85 or 100.

6. The polypeptide according to any one of the preceding embodiments, wherein the FG loop comprises an amino acid sequence selected from the group consisting of:

(a) SEQ ID NO: 8, 21, 34, 47, 60, 73, 86, 101, 129, 156, 183, 210, 237, 264, 291 or 318; and

(b) the FG loop has 1, 2 or 3 amino acid substitutions, insertions or deletions relative to the FG loop of SEQ ID NO: 8, 21, 34, 47, 60, 73, 86, 101, 129, 156, 183, 210, 237, 264, 291 or 318.

7. The polypeptide of any of the preceding embodiments, wherein the BC loop comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 19, 32, 45, 58, 71, 84 or 99; the DE loop comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 7, 20, 33, 46, 59, 72, 85 and 100; and the FG loop comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 21, 34, 47, 60, 73, 86, 101, 129, 156, 183, 210, 237, 264, 291 and 318, and wherein optionally, the BC, DE and/or FG loops comprise 1, 2 or 3 amino acid substitutions.

8. The polypeptide according to any one of the preceding embodiments, wherein:

(a) the BC, DE and FG loops comprise SEQ ID NOs: 6, 7 and 8, respectively;

(b) at least one of the BC, DE and FG loops comprises 1, 2 or 3 amino acid substitutions relative to the corresponding BC, DE and FG loops of SEQ ID NOs: 6, 7 and 8;

(c) the BC, DE and FG loops comprise SEQ ID NOs: 19, 20 and 21, respectively;

(d) at least one of the BC, DE and FG loops comprises 1, 2 or 3 amino acid substitutions relative to the corresponding BC, DE and FG loops of SEQ ID NOs: 19, 20 and 22;

(e) the BC, DE and FG loops comprise SEQ ID NOs: 32, 33 and 34, respectively;

(f) at least one of the BC, DE and FG loops comprises 1, 2 or 3 amino acid substitutions relative to the corresponding BC, DE and FG loops of SEQ ID NOs: 32, 33 and 34;

(g) the BC, DE and FG loops comprise SEQ ID NOs: 45, 46 and 47, respectively;

(h) at least one of the BC, DE and FG loops comprises 1, 2 or 3 amino acid substitutions relative to the corresponding BC, DE and FG loops of SEQ ID NOs: 45, 46 and 47;

(i) the BC, DE and FG loops comprise SEQ ID NOs: 58, 59 and 60, respectively;

(j) at least one of the BC, DE and FG loops comprises 1, 2 or 3 amino acid substitutions relative to the corresponding BC, DE and FG loops of SEQ ID NOs: 58, 59 and 60;

(k) the BC, DE and FG loops comprise SEQ ID NOs: 71, 72 and 73, respectively;

(l) at least one of the BC, DE and FG loops comprises 1, 2 or 3 amino acid substitutions relative to the corresponding BC, DE and FG loops of SEQ ID NOs: 71, 72 and 73;

(m) the BC, DE and FG loops comprise SEQ ID NOs: 84, 85 and 86, respectively;

(n) at least one of the BC, DE and FG loops comprises 1, 2 or 3 amino acid substitutions relative to the corresponding BC, DE and FG loops of SEQ ID NOs: 84, 85 and 86;

(o) the BC, DE and FG loops comprise SEQ ID NOs: 99, 100 and 101, respectively;

(p) at least one of the BC, DE and FG loops comprises 1, 2 or 3 amino acid substitutions relative to the corresponding BC, DE and FG loops of SEQ ID NOs: 99, 100 and 101.

9. The polypeptide of any one of the preceding embodiments, wherein the FBS comprises the amino acid sequence set forth in SEQ ID NO: 3, wherein the BC, DE and FG loops are represented by (X) _v , (X) _x and (X) _z , respectively, and

(a) comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the BC, DE and FG loop sequences set forth in SEQ ID NOs: 6, 7 and 8, respectively;

(b) comprising BC, DE and FG loops having the amino acid sequences of SEQ ID NOs: 6, 7 and 8, respectively;

(c) comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the BC, DE and FG loop sequences set forth in SEQ ID NOs: 19, 20 and 21, respectively;

(d) comprising BC, DE and FG loops having the amino acid sequences of SEQ ID NOs: 19, 20 and 21, respectively;

(e) comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the BC, DE and FG loop sequences set forth in SEQ ID NOs: 32, 33 and 34, respectively;

(f) comprising BC, DE and FG loops having the amino acid sequences of SEQ ID NOs: 32, 33 and 34;

(g) comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the BC, DE and FG loop sequences set forth in SEQ ID NOs: 45, 46 and 47, respectively;

(h) comprising BC, DE and FG loops having the amino acid sequences of SEQ ID NOs: 45, 46 and 47;

(i) comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the BC, DE and FG loop sequences set forth in SEQ ID NOs: 58, 59 and 60, respectively;

(j) comprising BC, DE and FG loops having the amino acid sequences of SEQ ID NOs: 58, 59 and 60;

(k) comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the BC, DE and FG loop sequences set forth in SEQ ID NOs: 71, 72 and 73, respectively;

(1) comprising BC, DE and FG loops having the amino acid sequences of SEQ ID NOs: 71, 72 and 73;

(m) comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the BC, DE and FG loop sequences set forth in SEQ ID NOs: 84, 85 and 86, respectively;

(n) comprising BC, DE and FG loops having the amino acid sequences of SEQ ID NOs: 84, 85 and 86;

(o) comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the BC, DE and FG loop sequences set forth in SEQ ID NOs: 99, 100 and 101, respectively;

(p) comprising BC, DE and FG loops having the amino acid sequences of SEQ ID NOs: 99, 100 and 101;

(q) comprising BC, DE and FG loops having the amino acid sequences of SEQ ID NOs: 99, 100 and 129;

(r) comprising BC, DE and FG loops having the amino acid sequences of SEQ ID NOs: 99, 100 and 156;

(s) comprising BC, DE and FG loops having the amino acid sequences of SEQ ID NOs: 99, 100 and 183;

(t) comprising BC, DE and FG loops having the amino acid sequences of SEQ ID NOs: 99, 100 and 210;

(u) comprising BC, DE and FG loops having the amino acid sequences of SEQ ID NOs: 99, 100 and 237;

(v) comprising BC, DE and FG loops having the amino acid sequences of SEQ ID NOs: 99, 100 and 264;

(w) comprising BC, DE and FG loops having the amino acid sequences of SEQ ID NOs: 99, 100 and 264; or

(x) comprising BC, DE and FG loops having the amino acid sequences of SEQ ID NOs: 99, 100 and 318.

10. The polypeptide of any of the preceding embodiments, wherein the FBS comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the non-BC, DE and FG loop regions of SEQ ID NO:3, 5, 18, 31, 44, 57, 70, 83 and 98.

11. The polypeptide of any of the preceding embodiments, wherein the FBS comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to any of SEQ ID NOs: 5, 18, 31, 44, 57, 70, 83 and 98.

12. The polypeptide of any of the preceding embodiments, wherein the FBS comprises an amino acid sequence that is at least 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of any one of SEQ ID NO: 5, 9-18, 22-31, 35-44, 48-57, 61-70, 74-83, 87-98, 102-128, 130-155, 157-182, 184-209, 211-236, 238-263, 265-290, 292-317 or 319-343.

13. The polypeptide of any of the preceding embodiments, wherein the FBS comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 98, 102-128, 129-155, 157-182, 184-209, 211-236, 238-263, 265-290, 292-317 and 319-343.

14. The polypeptide of any one of the preceding embodiments, wherein the FBS comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 18, 31, 44, 57, 70, 83, 98, 128, 155, 182, 209, 209, 236, 263, 290 and 317.

15. The polypeptide of any of the preceding embodiments, wherein the FBS comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 9-18, 22-31, 35-44, 48-57, 61-70, 74-83, 87-98, 102-128, 130-155, 157-182, 184-209, 211-236, 238-263, 265-290, 292-317 and 319-343.

16. The polypeptide of any one of the preceding embodiments, wherein the FBS comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 98, 102-128, 129-155, 157-182, 184-209, 211-236, 238-263, 265-290, 292-317 and 319-343.

17. The polypeptide according to any one of the preceding embodiments, wherein the FBS competes for binding to glypican 3 with a FBS comprising the amino acid sequence of SEQ ID NO:98.

18. The polypeptide according to any one of the preceding embodiments, wherein the FBS binds to a region within Glypican 3 comprising 10-20 amino acid residues of HQVSFF (SEQ ID NO: 209).

19. The polypeptide according to any one of the preceding embodiments, wherein the FBS binds to a region within Glypican 3 comprising 10-20 amino acid residues of EQLLQSASM (SEQ ID NO: 210).

20. The polypeptide according to any one of the preceding embodiments, wherein the FBS binds to a discontinuous Adnectin site within Glypican 3 comprising HQVSFF (SEQ ID NO: 209) and EQLLQSASM (SEQ ID NO: 210).

21. The polypeptide of any of the preceding embodiments, further comprising a heterologous protein.

22. The polypeptide of any of the preceding embodiments, further comprising one or more pharmacokinetic (PK) moieties selected from the group consisting of polyethylene glycol, sialic acid, Fc, Fc fragment, transferrin, serum albumin, serum albumin binding protein, and serum immunoglobulin binding protein.

23. The polypeptide according to any of the preceding embodiments, wherein the C-terminus of the FBS is linked to a module consisting of the _amino acid sequence _PmXn , wherein P is proline, X is any amino acid, m is an integer of at least 1, and n is 0 or an integer of at least 1.

24. The polypeptide of embodiment 23, wherein m is 1 or 2, and n is an integer from 1 to 10.

25. The polypeptide of embodiment 23 or 24, wherein the module comprises cysteine.

26. The polypeptide of embodiment 25, wherein the module consists of the amino acid sequence _PmCXn , wherein C is cysteine and each _X is independently any amino acid.

27. The polypeptide of embodiment 25, wherein the module consists of the amino acid sequence _PmCXn1CXn2 , wherein each X is independently any amino acid, _n1 and _n2 are independently 0 or an integer of at least 1 _.

28. The polypeptide of embodiment 28, wherein _n1 and _n2 are independently integers of 1-5.

29. The polypeptide of embodiment 23, wherein the module is selected from the group consisting of PI, PC, PID, PIE, PIDK (SEQ ID NO: 382), PIEK (SEQ ID NO: 383), PIDKP (SEQ ID NO: 384), PIEKP (SEQ ID NO: 385), PIDKPS (SEQ ID NO: 386), PIEKPS (SEQ ID NO: 387), PIDKPC (SEQ ID NO: 388), PIEKPC (SEQ ID NO: 389), PIDKPSQ (SEQ ID NO: 390), PIEKPSQ (SEQ ID NO: 391), PIDKPCQ (SEQ ID NO: 392), PIEKPCQ (SEQ ID NO: 393), PHHHHHH (SEQ ID NO: 394) and PCHHHHHH (SEQ ID NO: 395).

30. The polypeptide of embodiment 26, wherein the module is PC or PPC.

31. The polypeptide of embodiment 27, wherein the module is selected from the group consisting of PCGC (SEQ ID NO:412), PCPC (SEQ ID NO:413), PCGSGC (SEQ ID NO:414), PCPPPC (SEQ ID NO:415), PCPPPPPC (SEQ ID NO:416), PCGSGSGC (SEQ ID NO:417), PCHHHHHC (SEQ ID NO:418), PCCHHHHHH (SEQ ID NO:419), PCGCHHHHHH (SEQ ID NO:420), PCPCHHHHHH (SEQ ID NO:421), PCGSGCHHHHHH (SEQ ID NO:422), PCPPPCHHHHHH (SEQ ID NO:423), PCPPPPPHHHHHH (SEQ ID NO:424), and PCGSGSGCHHHHHH (SEQ ID NO:425).

32. The polypeptide of embodiment 31, wherein the module is PCPPPPPC (SEQ ID NO: 416).

33. The polypeptide of any one of embodiments 25-32, wherein the cysteine in the C-terminal module is conjugated to a heterologous module.

34. The polypeptide of embodiment 33, wherein the heterologous moiety is a detectable moiety.

35. The polypeptide of embodiment 33, wherein the heterologous moiety is a drug moiety, and the FBS and the drug moiety form a FBS-drug conjugate.

36. A FBS-drug conjugate comprising the FBS moiety of any one of embodiments 1-31, wherein the drug moiety is conjugated to the FBS moiety via a linker.

37. The FBS-drug conjugate of embodiment 36, wherein the linker is a hydrazone, a thioether, an ester, a disulfide bond, or a peptide-containing linker.

38. The FBS-drug conjugate of embodiment 37, wherein the linker is a peptidyl linker.

39. The FBS-drug conjugate of embodiment 38, wherein the peptidyl linker is Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val (SEQ ID NO: 467), Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, or Glu.

40. The FBS-drug conjugate of any one of embodiments 36-39, wherein the drug moiety is a cytotoxic drug.

41. The FBS-drug conjugate of embodiment 40, wherein the cytotoxic drug is selected from the group consisting of:

(a) Enediynes, such as calicheamicin and uncialamycin;

(b) tubulysin;

(c) DNA alkylating agents, such as CC-1065 analogs and duocarmycin;

(d) Epothilones;

(e) Auristatin;

(f) Pyrrolobenzodiazepine (PBD) dimers;

(g) Maytansinoids, such as DM1 and DM4

and their analogs and derivatives.

42. The FBS-drug conjugate of embodiment 41, wherein the drug moiety is:

43. The FBS-drug conjugate of any one of embodiments 36-41, wherein the drug moiety is a synthetic tubulysin analog having a structure of formula (II):

44. The FBS-drug conjugate of any one of embodiments 36-43, wherein the FBS and drug moiety are conjugated with a linker moiety having a structure of formula (III):

45. The FBS-drug conjugate of embodiment 44, wherein the drug-module-linker has the structure of formula (IV):

The maleimide group reacts with the sulfhydryl group of cysteine of FBS, thereby forming a thioether bond between the drug-moiety-linker and FBS.

46. The FBS-drug conjugate of any one of embodiments 36-45, wherein the FBS module contains a C-terminal module comprising cysteine.

47. The FBS-drug conjugate of embodiment 46, wherein the C-terminal module consists of the amino acid sequence _PmCXn , wherein C is cysteine, each X is independently any amino acid, m is an integer of at least 1, and _n is 0 or an integer of at least 1.

48. The FBS-drug conjugate of embodiment 47, wherein m is 1 or 2, and n is an integer from 1 to 10.

49. The FBS _- drug conjugate of embodiment 46, wherein the module consists of the amino acid sequence _PmCXn1CXn2 , wherein each X is independently any amino acid, _n1 and _n2 are independently 0 or an integer of at least 1.

50. The FBS-drug conjugate of embodiment 49, wherein _n1 and _n2 are independently integers of 1-5.

51. The FBS-drug conjugate of embodiment 47, wherein the C-terminal module is PC or PPC.

52. The FBS-drug conjugate of embodiment 49, wherein the module is selected from the group consisting of PCGC (SEQ ID NO: 412), PCPC (SEQ ID NO: 413), PCGSGC (SEQ ID NO: 414), PCPPPC (SEQ ID NO: 415), PCPPPPPC (SEQ ID NO: 416), PCGSGSGC (SEQ ID NO: 417), PCHHHHHC (SEQ ID NO: 418), PCCHHHHHH (SEQID NO: 419), PCGCHHHHHH (SEQ ID NO: 420), PCPCHHHHHH (SEQ ID NO: 421), PCGSGCHHHHHH (SEQ ID NO: 422), PCPPPCHHHHHH (SEQ ID NO: 423), PCPPPPPHHHHHH (SEQ ID NO: 424), and PCGSGSGCHHHHHH (SEQ ID NO: 425).

53. The FBS-drug conjugate of embodiment 52, wherein the module is PCPPPPPC (SEQ ID NO: 16).

54. A FBS-drug conjugate having a structure represented by formula (I),

wherein m is 1, 2, 3 or 4, and Adx is an Adnectin that specifically binds to human GPC3 with a _KD of 1 μM or less, and wherein the sulfur atom linked to "Adx" is the sulfur atom of the thiol group of cysteine of the Adnectin.

55. The FBS-drug conjugate of embodiment 54, wherein Adx is a ^human10Fn3 domain, wherein,

(a) the BC, DE and FG loops comprise SEQ ID NOs: 6, 7 and 8, respectively;

(b) the BC, DE and FG loops comprise SEQ ID NOs: 19, 20 and 21, respectively;

(c) the BC, DE and FG loops comprise SEQ ID NOs: 32, 33 and 34, respectively;

(d) the BC, DE and FG loops comprise SEQ ID NOs: 45, 46 and 47, respectively;

(e) the BC, DE and FG loops comprise SEQ ID NOs: 58, 59 and 60, respectively;

(f) the BC, DE and FG loops comprise SEQ ID NOs: 71, 72 and 73, respectively;

(g) the BC, DE and FG loops comprise SEQ ID NOs: 84, 85 and 86, respectively;

(h) the BC, DE and FG loops comprise SEQ ID NOs: 99, 100 and 101, respectively;

(i) the BC, DE and FG loops comprise SEQ ID NOs: 99, 100 and 129, respectively;

(j) the BC, DE and FG loops comprise SEQ ID NOs: 99, 100 and 156, respectively;

(k) the BC, DE and FG loops comprise SEQ ID NOs: 99, 100 and 183, respectively;

(l) the BC, DE and FG loops comprise SEQ ID NOs: 99, 100 and 210, respectively;

(m) the BC, DE and FG loops comprise SEQ ID NOs: 99, 100 and 237, respectively;

(n) the BC, DE and FG loops comprise SEQ ID NOs: 99, 100 and 264, respectively;

(o) the BC, DE and FG loops comprise SEQ ID NOs: 99, 100 and 264, respectively; or

(p) the BC, DE and FG loops comprise SEQ ID NOs: 99, 100 and 318, respectively.

Furthermore, the ¹⁰ Fn3 domain comprises a C _- terminal module consisting of the amino acid sequence _PmCXn or _PmCXn1CXn2 , wherein each X is independently any amino acid, and _{n1 and n2} are independently 0 or an integer of at least 1.

56. The FBS-drug conjugate of embodiment 55, wherein the C-terminal module is PC or PPC, PCGC (SEQ ID NO: 412), PCPC (SEQ ID NO: 413), PCGSGC (SEQ ID NO: 414), PCPPPC (SEQ ID NO: 415), PCPPPPPC (SEQ ID NO: 416), PCGSGSGC (SEQ ID NO: 417), PCHHHHHC (SEQ ID NO: 418), PCCHHHHHH (SEQ ID NO: 419), PCGCHHHHHH (SEQ ID NO: 420), PCPCHHHHHH (SEQ ID NO: 421), PCGSGCHHHHHH (SEQ ID NO: 422), PCPPPCHHHHHH (SEQ ID NO: 423), PCPPPPPHHHHHH (SEQ ID NO: 424) or PCGSGSGCHHHHHH (SEQ ID NO: 425).

57. The FBS-drug conjugate of embodiment 52, wherein the module is PC or PCPPPPPC (SEQ ID NO: 16).

58. The FBS-drug conjugate of embodiment 54, wherein Adx comprises an amino acid sequence selected from SEQ ID NOs: 12-17, 24-30, 38-43, 51-56, 64-69, 77-82, 90-97, 110-127, 137-154, 164-181, 191-208, 218-235, 245-262, 272-289, 299-316 and 326-343.

59. The FBS-drug conjugate of embodiment 54, wherein Adx comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 110-127, 137-154, 164-181, 191-208, 218-235, 245-262, 272-289, 299-316 and 326-343.

60. The FBS-conjugate of embodiment 54, wherein Adx comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 110-127.

61. A pharmaceutical composition comprising the polypeptide of any one of embodiments 1-35 and a pharmaceutically acceptable carrier.

62. A pharmaceutical composition comprising the FBS-drug conjugate of any one of embodiments 36-60.

63. The composition of embodiment 61 or 62, wherein the composition is substantially free of endotoxin.

64. An isolated nucleic acid molecule encoding the polypeptide of any one of embodiments 1-35.

65. An expression vector comprising a nucleotide sequence encoding the polypeptide of any one of claims 1-35.

66. A cell comprising a nucleic acid encoding the polypeptide of any one of embodiments 1-35.

67. A method of producing the polypeptide of any one of embodiments 1-35, the method comprising culturing the cell of claim 66 under conditions suitable for expression of the polypeptide, and purifying the polypeptide.

68. A method of reducing or inhibiting a Glypican 3 disease or disorder in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 61 or 62.

69. The method of embodiment 68, wherein the Glypican 3 disease or disorder is cancer.

70. The method of embodiment 69, wherein the cancer is hepatocellular carcinoma, melanoma, Wilms' tumor, hepatoblastoma, ovarian cancer, or squamous cell lung cancer.

71. A kit comprising the polypeptide of any one of embodiments 1-62, a FBS-drug conjugate or a pharmaceutical composition and instructions for use.

72. A method for detecting or measuring glypican 3 in a sample, comprising contacting the sample with the polypeptide of any one of embodiments 1-35, and detecting or measuring the binding of FBS to glypican 3.

73. An FBS-drug conjugate having a structure represented by formula (I),

wherein m is 1 and Adx is an Adnectin comprising the amino acid sequence of any one of SEQ ID NOs: 110-117 and 272-289; and wherein the sulfur atom linked to "Adx" is the sulfur atom of the thiol group of the C-terminal cysteine of the Adnectin.

74. A FBS-drug conjugate having a structure represented by formula (I),

wherein m is 2 and Adx is an Adnectin comprising the amino acid sequence of SEQ ID NOs: 119-126 and 281-288; and wherein the sulfur atom attached to "Adx" is the sulfur atom of the thiol group of each of the two C-terminal cysteines of the Adnectin.

75. A FBS-drug conjugate having a structure represented by formula (VI),

The sulfur atom bonded to cysteine is the sulfur atom of the sulfhydryl group of cysteine.

76. A FBS-drug conjugate having a structure represented by formula (VII),

The sulfur atom bonded to cysteine is the sulfur atom of the sulfhydryl group of cysteine.

References

The contents of all drawings and all references, Genbank sequences, websites, patents and published patent applications cited throughout this application are expressly incorporated herein by reference, to the same extent as if written in whole or in part in this document. In particular, the contents of PCT/US2015/021466 are incorporated herein by reference.

The present invention will now be described with reference to the following examples, which are illustrative only and are not intended to limit the present invention. Although the present invention has been described in detail with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications may be made thereto without departing from the spirit and scope of the present invention.

Example

Example 1: Selection, expression and purification of anti-glypican 3 binding Adnectin

GPC3-binding Adnectins are isolated from an Adnectin library screened with GPC3 protein, or clones identified from the library are obtained by PROfusion affinity maturation. Detailed descriptions of RNA-protein technology and fibronectin-based scaffold protein library screening methods are provided in Szostak et al., U.S. Patent Nos. 6,258,558; 6,261,804; 6,214,553; 6,281,344; 6,207,446; 6,518,018; PCT Publication Nos. WO 00/34784; WO 01/64942; WO 02/032925; and Roberts et al., Proc Natl. Acad. Sci., 94: 12297-12302 (1997), which are incorporated herein by reference.

The amino acid and nucleotide sequences of seven adnectins with good binding and biophysical properties are provided below:

ADX_4578_F03

MGVSDVPRDLEVVAATPTSLLISWHPPHPNIVSYHIYYGETGGNSPVQEFTVEGSKSTAKISGLKPGVDYTITVYAVAPEIEKYYQIWINYRTEGSGS*(SEQ ID NO:10)

ATGGGAGTTTCTGATGTGCCGCGCGACTTGGAAGTGGTTGCCGCCACCCCCACCAGCCTGCTGATCTCTTGGCATCCGCCGCATCCGAACATCGTTTCTTACCATATCTACTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGGAAGGTTCTAAATCTACTGCTAAAATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTACGCTGTTGCTCCGGAAATCGAAAAATACTACCAG ATTTGGATTAATTACCGCACAGAAGGCAGCGGTTCCTAA(SEQ ID NO:452)

ADX_4578_H08

MGVSDVPRDLEVVAATPTSLLISWSGYDYGDSYYRITYGETGGNSPVQEFTVPDGSNTATISGLKPGVDYTITVYAVEAYGKGYTRYTPISINYRTEIDKPSQ*(SEQ ID NO:23)

ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATCAGCTGGTCTGGTTACGACTACGGTGACTCTTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGACGGTTCTAACACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAAGCTTACGGTAAAGGTTACACT CGTTACACTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGTAA(SEQ ID NO:452)

ADX_4578_B06

MGVSDVPRDLEVVAATPTSLLISWFPDRYVYYITYGETGGNSPVQEFTVEGHKQTAYISGLKPGVDYTITVYAIYYYPDDFQGYPQPISINYRTEGSGS*(SEQ ID NO:36)

ATGGGAGTTTCTGATGTGCCGCGCGACTTGGAAGTGGTTGCCGCCACCCCCACCAGCCTGCTGATCTCTTGGTTCCCGGACCGTTACGTTTACTACATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGGAAGGTCATAAACAGACTGCTTACATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTACGCTATCTACTACTACCCGGACGACTTCCAGGGTTACCCGCAGC CGATTTCTATTAATTACCGCACAGAAGGCAGCGGTTCCTAA(SEQ ID NO:454)

ADX_4606_F06

MGVSDVPRDLEVVAATPTSLLISWNSGHSGQYYRITYGETGGNSPVQEFTVPRYGYTATISGLKPGVDYTITVYAVAHSEASAPISINYRTEIDKPSQ*(SEQID NO:49)

ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATCAGCTGGAACTCTGGTCATTCTGGTCAGTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTCGTTACGGTTACACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGCTCATTCTGAAGCTTCTGCTCCAATT TCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGTAA(SEQ ID NO:455)

ADX_5273_C01

MGVSDVPRDLEVVAATPTSLLISWSDPYEEERYYRITYGETGGNSPVQEFTVPAFHTTATISGLKPGVDYTITVYAVTYKHKYAYYYPPISINYRTEIDKPSQ*(SEQ ID NO:62)

ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATCAGCTGGTCTGACCCGTACGAAGAAGAACGATATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGCTTTCCATACTACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTTACAAACATAAATACGCTTACTACT ACCCGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGTAA(SEQ ID NO:456)

ADX_5273_D01

MGVSDVPRDLEVVAATPTSLLISWEPSYKDDRYYRITYGETGGNSPVQEFTVPSFHQTATISGLKPGVDYTITVYAVTYEPDEYYFYYPISINYRTEIDKPSQ*(SEQ ID NO:75)

ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATCAGCTGGGAACCGTCTTACAAAGACGACCGATATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTTCTTTCCATCAGACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTTACGAACCGGACGAATACTACT TCTACTACCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGTAA(SEQ ID NO:457)

ADX_5274_

MGVSDVPRDLEVVAATPTSLLISWSGDYHPHRYYRITYGETGGNSPVQEFTVPGEHETATISGLKPGVDYTITVYAVTYDGEKADKYPPISINYRTEIDKPSQ*(SEQ ID NO:88)

ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATCAGCTGGTCTGGTGACTACCATCCGCATCGATATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGGTGAACATGAAACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTTACGACGGTGAAAAAGCTGA CAAATACCCGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGTAA(SEQ ID NO:458)

Using GPC3 positive CHO cell lines and HepG2 human tumor cell lines, the binding characteristics of 7 GPC3 binding Adnectins were determined by ELISA using recombinant GPC3 and flow cytometry. For flow cytometry experiments, CHO-K1 or CHO-Glypican 3 cells or HepG2 tumor cell lines were treated with ethylenediaminetetraacetic acid (Versene) and resuspended in FACS buffer (PBS 2.5% FBS). Adnectin diluted in FACS buffer was incubated with cells at 4°C for 1 hour. After washing once in FACS buffer, cells were incubated with 2 μg/ml of anti-His antibody and incubated at 4°C for 1 hour. After washing twice in FACS buffer, cells were resuspended in FIX buffer (2.5% formaldehyde in PBS). Analyzed with BBBiosciences FACS Canto.

The results of the ELISA experiments are shown in Table 3, and the results of exemplary flow cytometry are shown in Figures 3A-D. Table 2 also provides the aggregation scores of the Adnectins determined by size exclusion chromatography (SEC). None of these Adnectins aggregated significantly.

Table 2: ELISA and SEC Scoring of Human GPC3 Binding Adnectins

The C-terminus of ADX_5274_E01 was modified by inclusion of a C-terminal cysteine and a 6xHis tail to generate AdnectinADX_6561_A01:

MGVSDVPRDLEVVAATPTSLLISWSGDYHPHRYYRITYGETGGNSPVQEFTVPGEHETATISGLKPGVDYTITVYAVTYDGEKADKYPPISINYRTPCHHHHHH(SEQ ID NO:94)

Nucleic acids encoding ADX_6561_A01 were diversified by introducing a small number of substitutions at each nucleotide position encoding an amino acid residue in loops BC, DE or FG. The resulting library of Adnectin sequences related to ADX_6561_A01 was then selected in vitro for binding to human GPC3 under high stringency conditions by PROfusion (mRNA display). After selection was complete, the enriched clones were sequenced, expressed as HTPP format, and further analyzed.

The selection identified Adnectin ADX_6077_F02 as binding to human GPC3 with high affinity. The amino acid sequence of ADX_6077_F02 and the nucleotide sequence encoding it are as follows:

MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDGEKAATDWSISINYRTPCHHHHHH (SEQ ID NO: 118; BC, DE and FG loops shown in bold)

ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATCAGCTGGTCTGATGACTACCATGCGCATCGATATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGGTGAACATGTGACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTTACGACGGTGAAAAGGCTGCCACA GATTGGTCAATTTCCATTAATTACCGCACACCGTGCCACCATCACCACCACCACTGA(SEQ ID NO:459)

The anti-GPC3 Adnectin was tested for binding to other glypican molecules and the results showed that ADX_6077_F02 specifically bound to human GPC3 and did not cross-react with other human glypicans GPC1, GPC2, GPC5 and GPC6.

Example 2: Adnectin with C-terminal cysteine for conjugation of drug moieties

To prepare an Adnectin linked to a drug moiety, the Adnectin was modified at its C-terminus to comprise one of the following C-terminal amino acid sequences: NYRTPC (SEQ ID NO: 466; for forming a DAR1 Adnectin, i.e., an Adnectin with a single cysteine in the linker for linkage to a single drug moiety); NYRTPCC (SEQ ID NO: 467; for forming a DAR2 Adnectin, i.e., an Adnectin with two cysteines in the linker for linkage to two drug moieties, one at each); NYRTPCHHHHHH (SEQ ID NO: 468; for forming a DAR1 Adnectin with a 6xHis tail) and NYRTPCPPPPPCHHHHHH (SEQ ID NO: 469; for forming a DAR2 Adnectin with a 6xHis tail).

To prevent the generation of dislufide-linked dimers of unconjugated Adnectins containing one or more cysteine residues, the cysteine residues of the Adnectins were carboxymethylated as follows: the Adnectin solution was treated with a reducing agent (5 mM DTT or 5 mM TCEP) and incubated at room temperature for 30 minutes. Iodoacetamide (500 mM, Sigma P/N A3221-10VL) was added to a final concentration of 50 mM. The samples were incubated at room temperature in the dark for 1 hour. The samples were then dialyzed in PBS or sodium acetate buffer.

Example 3: Production of GPC3-Adnectin Drug Conjugate (GPC3-AdxDC)

Production of Adnectin, such as GPC3 Adnectin: A nucleic acid encoding an Adnectin, such as a nucleic acid encoding a gene having the amino acid sequence

The protein of MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDGEKAATDWSISINYRTPCHHHHHH (SEQ ID NO: 118; ADX_6077_F02) (SEQ ID NO: 459) was cloned into the pET9d (EMD Biosciences, San Diego, CA) vector and expressed in E. coli BL21 DE3 pLys-S cells. A 20 ml inoculum culture (generated from a single plated colony) was used to inoculate 1 liter of Magic Media E. coli expression medium (Invitrogen, catalog number K6803A/B) containing 50 μg/ml kanamycin in a 2.5 liter Ultra Yield flask (Thomson Instruments Co. P/N 931136-B). The culture was grown at 37°C for 6 hours, then shaken at 20°C for 18 hours at 225RPM. After the incubation period, the culture was harvested by centrifugation at 4°C for 30 minutes with ≥10,000g. The cell pellet was frozen at -80°C. The cell pellet was melted and resuspended in 25mL lysis buffer (20mM sodium phosphate, 500mM sodium chloride, 5mM dithiothreitol, 1x EDTA-free Complete ^TM protease inhibitor mixture (Roche)) using Ultra-Turrax homogenizer (IKA-Works) on ice. Cell lysis was achieved by high pressure homogenization (≥18,000psi) using M-110P microfluidizer (Microfluidics). Insoluble parts were separated and discarded by centrifugation at 4°C for 30 minutes with 23,300g. The soluble part was filtered with a 0.2 micron vacuum filter. The supernatant of filtration is loaded onto the Histrap post (GE Healthcare P/N 17-5248-02) balanced with 20mM sodium phosphate/500mM sodium chloride (pH 7.4)+5mM DTT buffer.After loading, the post is washed with 10 CV of equilibrium buffer, then washed with 10 CV of 40mM imidazoles in equilibrium buffer, then washed with 10 CV of 2.0M sodium chloride in PBS.The protein bound by 500mM imidazole elution in 20mM sodium phosphate/500mM sodium chloride (pH 7.4)+5mM DTT is used.The eluent from HisTrap post and 50mM sodium acetate/10mM sodium chloride (pH 5.5) buffer exchange are used using G25 gel filtration chromatography.Then the sample is applied to a cation exchange chromatographic column (SP HP, GE Healthcare 17-1152-01). The bound protein was eluted with an increasing sodium chloride concentration gradient in 50 mM sodium acetate (pH 5.5) buffer. The fractions were pooled and used for conjugation with tubulysin.

Generation of tubulysin analog-linker : The tubulysin analog-linker compound having the structure of formula (IV) was generated as described in US Pat. No. 8,394,922 (incorporated herein by reference).

Adnectin-drug conjugation: Conjugation of tubulysin analog-linker to Adnectin containing a C-terminal cysteine was performed as follows:

The adnectin samples to be conjugated to tubulysin analogs were treated with 5mM TCEP and incubated at room temperature for about 1 hour. TCEP was removed using a G25 gel filtration column (GE Healthcare) equilibrated with 50mM NaOAc/10mM NaCl (pH 5.5). The tubulysin analogs were dissolved in 100% DMSO and added to a final concentration of 5x molar, and the reaction was incubated at room temperature for 2 hours and then overnight at 4°C. To remove unreacted tubulysin analogs, the reaction mixture was reapplied to the SP cation exchange column as described above.

Using the same approach described above, an Adnectin containing two cysteine residues near the C-terminus (eg, GPC3 Adnectin) is conjugated to two molecules of a tubulysin analog to generate a DAR2 (drug-to-Adnectin ratio of 2) Adnectin.

Protein concentrations were determined using a Nanodrop 8000 spectrophotometer (Thermo Scientific). The molecular weights of conjugated and unconjugated Adnectins were determined by LC mass spectrometry using an Agilent Technologies 6540 UHD Accurate Mass Q-ToF LC-MS equipped with a Zorbax C8RRHD column.

Using these techniques for expressing, purifying, conjugating, and alkylating Adnectins, the Adnectins and Adnectin-drug conjugates listed in Table 3 were prepared.

Table 3 - Control and anti-glypican 3 binding Adnectins

Example 4: In vitro characterization of anti-GPC3 Adnectin and GPC3-AdxDC

Size Exclusion Chromatography: Standard size exclusion chromatography (SEC) was performed on the candidate Adnectins generated from the mid-scale process. SEC of the mid-scale material was performed using a Superdex 200 10/30 or a Superdex 75 10/30 column (GE Healthcare) on an Agilent 1100 or 1200 HPLC system with UV detection at A214nm and A280nm and fluorescence detection (excitation 280nm, emission 350nm). A buffer of 100mM sodium sulfate/100mM sodium phosphate/150mM sodium chloride at pH 6.8 was used at an appropriate flow rate for the SEC column employed. Molecular weight calibration was performed using gel filtration standards (Bio-Rad Laboratories, Hercules, CA).

Thermal stability: Thermal scanning fluorescence (TSF) analysis of HTPP Adnectins was performed to screen them by relative thermal stability. Samples were standardized to 0.2 mg/ml in PBS. 1 μl of Sypro Orange dye was diluted 1:40 with PBS, added to 25 μl of each sample, and the plate was sealed with a transparent 96-well microplate sealant. The samples were scanned using a BioRad RT-PCR machine by gradually increasing the temperature from 25°C to 95°C at a rate of 2 degrees per minute. The data were analyzed using BioRad CFX manager 2.0 software. The values obtained by TSF have been shown to correlate well with the Tm values obtained by DSC in the melting range of 40°C to 70°C. This is considered to be an acceptable working range for this technology. ND ("no data") results are obtained when the slope of the transition curve is too small to allow its derivative peak (the rate of change of fluorescence over time) to be distinguished from the noise. "ND" results cannot be interpreted as an indication of thermal stability. Differential scanning calorimetry (DSC) analysis of dialyzed HTPP'd and mid-scale Adnectins was performed to determine their corresponding _Tm 's. 0.5 mg/ml solutions were scanned in a VP-capillary differential scanning calorimeter (GE Microcal) at 70 psi pressure by ramping the temperature from 15°C to 110°C at a rate of 1 degree per minute. Data were analyzed relative to the appropriate buffer control run using best fit using Origin Software (OriginLab Corp).

SPR affinity measurements: Surface plasmon resonance (SPR) was performed using a Biacore T100 instrument (GE Healthcare) to calculate the dissociation rate ( _kd ) and binding affinity of α-GPC3 adnectin and tubulysin-conjugated AdxDC. Following the manufacturer's amine coupling protocol (GE Healthcare), recombinant human (aa 1-559) and mouse (aa 25-557) glypican 3 proteins (R&D Systems) were diluted to 10 μg/ml in 10 mM sodium acetate (pH 4.5) and immobilized individually on the active flow cell of a CM5 biosensor, aiming for an immobilization density of approximately 1000 RU per flow cell for each protein. SPR experiments were performed at 37°C using HBS-P+ (10 mM HEPES, 150 mM NaCl, 0.05% (v/v) surfactant P20, pH 7.4) running buffer (GE Healthcare). For affinity measurements, a concentration series of α-GPC3 adnectin and AdxDC was prepared from 200 to 1.56 nM in running buffer and injected into human and mouse GPC3 biosensor flow cells at 30 μl/min. For off-rate measurements, a single 200 nM concentration of adnectin/AdxDC was injected using the same conditions. Between assay cycles, a single 30 sec injection of 10 mM glycine (pH 1.7) was used to remove bound adnectin and regenerate the GPC ₃ surface.

Rate constants _ka ( _kon ) and _kd ( _koff ) were derived from reference-subtracted sensorgrams fitted to a 1:1 binding model in Biacore T100 Evaluation Software 2.0.4 (GE Healthcare). The affinity constant _KD was calculated from the ratio of the rate constants _kd / _ka .

Cell binding assay: The binding of GPC3 adnectin to huGPC3 positive cells Huh7 was assessed by flow cytometry essentially as follows. Huh7 cancer cells were grown in DMEM medium containing 10% FBS. Cells were harvested using Versene (an EDTA cell dissociation solution) from Lonza catalog number 17-711E. Tumor cells (1E5 cells/reaction) were suspended in FACS buffer (PBS, 1% BSA, 0.05% sodium azide) and mixed with serial dilutions of AdxDC on ice for one hour. Cells were washed three times with FACS buffer, and bound AdxDC was detected with an in-house anti-scaffold monoclonal Ab and a PE-conjugated antibody from (RnD Systems) catalog number NL007, and read on a flow cytometer. Data analysis was performed using FlowJo software, and the EC50 of 50% maximum binding was determined using PRISM ^TM software (GraphPad Software, La Jolla, CA, USA, version 5.0).

Cell growth inhibition assay: The dose-dependent inhibitory effect of AdxDC on Hep3B, Huh7 and HepG2 cell proliferation was evaluated using the ³ H thymidine assay, where the inhibition of ³ H thymidine incorporation indicates the inhibition of test cell line proliferation. Human tumor cell lines were obtained from the American Type Culture Collection (ATCC) (P.O. Box 1549, Manassas, VA 20108, USA) and cultured according to the instructions from ATCC. Cells were seeded in 96-well plates at 1.25 x 10 ⁴ cells/well, and 1:3 serial dilutions of GPC3 AdxDC were added to the wells. The plates were allowed to incubate for 72 hours. In the last 24 hours of the total incubation period, the plates were pulsed with 1.0 μCi of ³ H-thymidine per well, harvested, and read on a Top Count scintillation counter (Packard Instruments, Meriden, CT). _EC50 values - the concentration of Adnectin drug conjugate that achieves 50% of maximal inhibition of cell proliferation - were determined using PRISM ^™ software, version 4.0 (GraphPad Software, La Jolla, CA, USA).

A summary of the in vitro properties of the AdxDC conjugates in the DAR1 and DAR2 formats is summarized in Table 4.

Table 4: In vitro characterization of GPC3-tubulin AdxDC

^alk - measured for alkylated (capped Adnectin); all other measurements for DAR1AdxDC (no PEG)

Example 5: Cell Binding of GPC3-AdxDC to Human Hep3B and H446 Tumor Cells

The binding of GPC3 AdxDC to human Hep3B hepatocellular carcinoma cells grown in MEM containing 10% FBS and to H446 small cell lung cancer cells grown in RPMI containing 10% FBS was evaluated by flow cytometry. Cells were harvested using a non-enzymatic cell dissociation solution Cellstripper from Mediatch (Corning: Manassas, VA 20109) catalog number 25-056-CL. Tumor cells (25,000/reaction) were suspended in FACS buffer (PBS + 5% FBS + 0.01% NaN ₃ ) and mixed with serially diluted AdxDC on ice for 1 hour. Cells were washed three times with FACS buffer, and bound AdxDC was detected with a His tag PE-conjugated antibody from R&D System catalog number IC050P and read on a flow cytometer. Data analysis was performed using FlowJo software, and the EC50 for 50% maximal binding was determined using PRISM ^™ software, version 5.0 (GraphPad Software, La Jolla, CA, USA).

The results shown in Figures 4A-D indicate that ADX_6077_F02AdxDC DAR1 and DAR2 bind to both types of human tumor cells.

Example 6: GPC3 AdxDC inhibits cell growth of Hep3B, H446 and HepG2 tumor cells

This example shows that GPC3 AdxDC DAR1 and DAR2 inhibit cell proliferation of Hep3B (Glypican 3 High) HCC cells, H446 (Glypican 3 Low) SCLC cells, and HepG2 tumor cells. Thymidine incorporation assays were performed as described above. The results shown in Figures 5A-B and 6A-B show that GPC3 AdxDC DAR1 and DAR2 inhibited cell growth of three different cell lines, but the control AdxDC adnectin conjugate did not inhibit the growth of these cells.

Example 7: Time course of cell surface binding assay of GPC3-AdxDC

To ensure maximum target engagement prior to internalization studies, the following binding assay was used to determine the binding of ADX_6077_F02DAR1 (i.e., with a "PC" end, but not conjugated) to GPC-3 positive cells Hep3B: In the Hep3B cell binding assay, AF-488 fluorescently labeled adnectin ADX_6077_F02 and negative control (NBC) ADX_6093_A01 were used. For binding analysis, Hep3B cells were plated into 384-well plates, incubated for 16 hours to allow cell adhesion, and then fixed with 2% formaldehyde. 100 nM of ADX_6077_F02 and ADX_6093_A01 were added to the cell plates and incubated at room temperature for 7 time points: 0 minutes, 10 minutes, 15 minutes, 20 minutes, 60 minutes, 120 minutes, and 180 minutes. After binding, cells were washed twice with phosphate-buffered saline (PBS), and total cellular fluorescence intensity per cell was measured using high-content analysis.

The results showed that ADX_6077_F02 exhibited rapid binding to the cell surface. Using 100 nM Adnectin, two hours was found to be sufficient to reach a binding plateau of greater than 95%.

Example 8: Kinetic internalization of GPC3-AdxDC

To quantify the internalization induced by anti-Glypican 3 adnectin, a high-content Alexa quenching assay was applied. Hep3B and H446 cells were seeded in 384-well plates and incubated at 37°C for 16 hours. Then 100 nM of AF-488 fluorescently labeled ADX_6077_F02DAR1 was added to the cell plate and incubated at 37°C for the indicated time before fixation and quenching. The internalized Adnectin was measured as the increase in fluorescence above the signal that could not be quenched. The total fluorescence from the "unquenched control" at each time point was monitored in parallel and used as an indicator of the amount of adnectin initially bound to the cells. Images of the cells were taken by Arrayscan to show the localization of the adnectin, and the images of the cells were used for quantification of cell fluorescence intensity.

Quantitative studies confirmed high expression levels of GPC3 receptor on Hep3B (approximately 1.1 x 10 ⁶ active binding copies/cell) and lower levels of GPC3 receptor on H446 cells (approximately 2.6 x 10 ⁵ active binding copies/cell).After fixation, total and intracellular FL were determined and used to measure internalization of Adnectin molecules.

The results of these assays showed that anti-GPC3 adnectin was internalized by Hep3B and H446 cells (Figure 7) at a moderate-slow rate (T1 _/2 >1 hour), and reached >90% internalization after 6 hours. As shown in Figure 8, at the 15 minute time point, most of the anti-GPC3 adnectin was membrane-bound, and at the 8 hour time point, most of the GPC3-Adectin signal was located inside the cells.

Example 9: In vivo pharmacokinetics of GPC3-AdxDC

The systemic exposure of anti-GPC3 AdxDC (DAR1) in mice was determined. According to the following experimental design, female NOD/SCID mice (13 weeks old) were intravenously administered high (240nmol/kg) and low (24nmol/kg) doses of single doses of GPC3 binding and non-binding control AdxDC (GPC3DAR1AdxDC and RGE AdxDC, respectively). The indicated blood sampling time points were continuous tail vein blood sampling using CPD anticoagulant (citrate-phosphate-dextrose solution, Sigma C7165). The plasma obtained from these blood samples was aliquoted and stored at -80°C until ready for analysis.

Table 5 - Dosing regimen for xenograft model

AdxDC plasma levels were analyzed using a mid-scale (MSD) ligand binding assay with two different formats. The MSD assay for total levels of conjugated and unconjugated Adnectin assays used to capture in-house produced anti-His monoclonal antibodies (at 4 μg/ml) and to detect pooled in-house produced rabbit anti-scaffold polyclonal antibodies diluted 1:10000 followed by goat anti-rabbit sulfotagged antibodies (at 1 μg/ml). The MSD assay for intact conjugated Adnectins used to capture in-house produced anti-His monoclonal antibodies (at 4 μg/ml) and to detect in-house produced sulfotagged mouse anti-tubullysin antibodies (at 1 μg/ml).

The results of this assay are summarized in Table 6 (non-compartmental Phoenix WinNonlin analysis, NCA model) and Figure 9 (anti-tubullysin MSD assay), which further demonstrate that AdxDC has a short exposure profile in mice.

Table 6 - Summary of pharmacokinetic parameters of GPC3 AdxDC

Example 10: Inhibition of tumor growth in a rodent xenograft model

The efficacy of the non-PEGylated GPC3-tubullysin drug conjugate was tested in CD1 mice and Fischer rats.

NOD-SCID and CD1 female mice (13 weeks old, from Charles River Laboratories, Wilmington, MA) and female Fischer rats (10 weeks old, from Charles River laboratories, Wilmington, MA) were housed in a temperature-controlled room with a reversed 12-hour light/dark cycle. Water and standard chow were available ad libitum. Animals used for safety studies were randomized and allocated between treatment groups, receiving either control or test AdxDC according to body weight (approximately 20-25 g).

Hep3B (human hepatocellular carcinoma) was maintained in culture using EMEM catalog number ATCC 30-2003 supplemented with 10% FBS (Thermo catalog number ATK-33398). For efficacy studies, xenografts were generated by subcutaneously implanting 100 μl of 5×10 ⁶ Hep3B cells (50% cell suspension, containing standard phenol red matrix gel (Phenol Red Matrigel, Corning catalog number 354234) in the right flank of NOD-SCID mice. To demonstrate in vivo efficacy, AdxDC was administered by intravenous injection with 50 mM NaOAc/150 mM NaCl/pH5.5 or phosphate buffered saline (PBS). Controls were treated with non-binding control AdxDC. Experimental animals (n=8 animals/group) were intravenously administered every three days for a total of 6 doses, with different AdxDC doses. Body weight measurements were recorded before and on the day of randomization, twice a week during treatment, and at the end of the study. Tumor growth was monitored twice a week using digital caliper measurements. Results were evaluated using a Student's t-test two-tailed paired analysis. A representative study design and results using a three-day dosing regimen are provided in Table 7 and Figure 10.

Table 7: Dosage regimen

Weekly dosing was evaluated in Hep3B xenografts.The results showed that QW administration of 0.1 μmol/kg of ADX_6077_F02-961DAR1 and DAR2 effectively inhibited HepG2 xenografts: _TV0 = 380-480 ^mm3 (Figure 11), _TV0 = 228-350 ^mm3 (Figure 12), and _TV0 = 514-673 ^mm3 (Figure 13).

In conclusion, weekly administration of GPC3 AdxDCs inhibited the growth of HCC tumor xenografts, and DAR1 and DAR2 GPC3 AdxDCs exhibited equivalent tumor growth inhibition, which was target-dependent. Additionally, prevention of tumor burden-induced weight loss was associated with the antitumor activity of GPC3 AdxDCs.

In the mouse safety study, CD1 mice were intravenously injected every other day at different doses for a total of 9 doses, with the highest dose reaching 0.5 μmol/kg of GPC3 and non-binding control AdxDC, which is 5 times the effective dose (0.1 μmol/kg). MTD was not identified in the CD1 mouse safety study. No renal toxicity was observed for any group at any dose or frequency. In CD1 mice, the half-life of AdxDC is approximately 20 minutes (MSD determination as described above). Until the scheduled autopsy, no weight loss was observed, and all mice survived the treatment. Serum chemistry and hematology evaluations were performed at intervals during dosing using Abaxis Veterinary Diagnostics instruments VETSCAN VS2 and HM5, respectively. No significant differences were observed in serum chemistry or hematology compared to baseline. At the end of the study, histopathological evaluations were performed by H&E staining of collected heart, liver, spleen and kidney tissues. No dose-limiting toxicity was observed in any of the evaluated tissues, and minimal/mild renal tubular epithelial neuropathy was observed in all groups.

In the Fischer rat safety study, the half-life of AdxDC was approximately 30 minutes (MSD determination as described above). At the most frequent dosing (every other day) at a dose of 0.36 μmol/kg, some dose-dependent tolerance and skeletal muscle degeneration were observed, with no changes in bone marrow or liver histopathology or cardiotoxicity. These findings were not observed when the same rat study was performed using weekly dosing of AdxDC over the same dose range.

Overall, excellent efficacy consistent with low systemic exposure was observed in both rodent species, despite a short plasma half-life and low off-target toxicity.

Example 11: Mapping of Adnectin binding sites on human GPC3 using HDX-MS.

Adnectin binding sites on human GPC3 (amino acid sequence shown in Figure 14) were evaluated using hydrogen-deuterium exchange mass spectrometry (HDX-MS). The hydrogen/deuterium exchange mass spectrometry (HDX-MS) method probes protein conformation and conformational dynamics in solution by monitoring the rate and extent of deuterium exchange in backbone amide hydrogens. The level of HDX depends on the solvent accessibility of backbone amide hydrogens as well as the conformation of the protein. The mass increase of a protein on HDX can be precisely measured by MS. When this technique is paired with enzymatic digestion, structural features at the peptide level can be obtained, allowing surface-exposed peptides to be distinguished from those that are internally folded or sequestered at the interface of a protein-protein complex. Typically, deuterium labeling and subsequent quenching experiments are performed, followed by online pepsin digestion, peptide separation, and MS analysis.

Prior to mapping the Adnectin binding site on human GPC3 recognized by ADX_6077_F02 by HDX-MS, a non-deuterated experiment was performed to generate a list of common peptic peptides of the GPC3 sample, achieving 87.4% sequence coverage of GPC3 (Figure 14). In this experiment, 10 mM phosphate buffer (pH 7.0) was used during the labeling step, followed by the addition of quenching buffer (200 mM phosphate buffer containing 4 M GdnCl and 0.4 M TCEP, pH 2.5, 1:1, v/v).

For Adnectin binding site mapping experiments, 5 μL of each sample (GPC3 or GPC3 with ADX_6077_F02) was mixed with 55 μL HDX labeling buffer (10 mM phosphate buffer in D2O, pD 7.0) to start the labeling reaction. The reactions were run for different times: 1 minute, 10 minutes, and 240 minutes. At the end of each labeling reaction period, the reaction was quenched by adding quenching buffer (1:1, v/v), and the quenched samples were injected into the Waters HDX-MS system for analysis. The observed deuterium uptake levels of common peptic peptides were monitored in the absence/presence of ADX_6077_F02 (Figures 14 and 15).

Experimental data obtained from HDX-MS measurements indicate that AADX_6077_F02 recognizes a discontinuous Adnectin binding site consisting of two peptide regions in human GPC3:

Region 1: HQVRSFF (amino acid residues 36-42 of GPC3); SEQ ID NO: 356

Region 2: EQLLQSASM (amino acid residues 90-98 of GPC3); SEQ ID NO: 346

Example 12: Generation of DG variants

Analysis of the amino acid sequence of ADX_6077_F02 indicated that the DG in the FG loop of this molecule may have a low risk of aspartate isomerization.

MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDG EKAATDWSISINYRTPCHHHHHH (SEQ ID NO:118)

Eight ADX_6077_F02 variants with mutations at the DG position were generated. The sequences of these mutants are summarized in Table 8.

Table 8: ADX_6077_F02 variants

Example 13: Biophysical Characterization of DG Variants

As described above, 100-150 mg of each of the eight mutants were prepared, purified and alkylated. 3 to 5 mg of each of the eight alkylated variants were subjected to SEC, DSC, GPC3 binding (SPR 1pt dissociation rate), MS and HIC at 1-3 mg/mL. The results are summarized in Table 9.

Table 9: Biophysical properties of the ADX_6077_F02DG variant

Six of the eight DG mutants exhibited a 3-5 fold increase _{in koff} compared to the parent adnectin and were monomeric and thermostable. The binding affinity of the alkylated GPC3 DG mutant adnectins to human and mouse GPC3 was further evaluated by Biacore T100 using HBS-P+ running buffer, with direct immobilized human and mouse GPC3 proteins [Hu (Fc 2,3) and Mu (Fc 4) GPC3-His (R&D Systems)] injected serially from 200-1.56 nM, 180 s association, 600 s dissociation. The data were fit to a 1:1 binding model in the BiaEvaluation software and are summarized in Table 10.

Table 10 - Binding kinetics of DG mutant Adnectins

The data demonstrated that these GPC3DG mutants had approximately 3-5 fold lower affinity for human and mouse GPC3 compared to the parental ADX_6077_F02. The difference in affinity was driven by a faster off-rate, while the on-rate was consistent with the parental adnectin (Figure 16).

Example 14: Evaluation of Cell Binding and Immunogenicity of DG Variants

The binding of DG variants for huGPC3 in DG GPC3 AdxDC mutants to Huh7 cancer cells cultured in DMEM medium containing 10% FBS was evaluated by flow cytometry. Cells were harvested using Versene (an EDTA cell dissociation solution) from Lonza catalog number 17-711E. Tumor cells (1E5 cells/reaction) were suspended in FACS buffer (PBS, 1% BSA, 0.05% sodium azide) and mixed with serial dilutions of AdxDC on ice for one hour. The cells were washed three times with FACS buffer, and the bound AdxDC was detected with an internal anti-scaffold monoclonal Ab and a PE-conjugated antibody from (RnD Systems) catalog number NL007, and read on a flow cytometer. Data analysis was performed using FlowJo software, and the EC50 of 50% maximum binding was determined using PRISM ^TM software (GraphPad Software, La Jolla, CA, USA, version 5.0).

For anti-His detection (of non-DG mutants), the same protocol was used except that an in-house generated APC-conjugated anti-His antibody was used.

The results shown in Table 11 indicate that the mutants had similar EC50 values as the parental Adnectin.

The potential of DG variants for huGPC3 to elicit an immune response in humans was also assessed using a human PBMC proliferation assay. In the presence of DG variants or controls, PBMCs from 40 donors with HLA class II haplotypes that closely match the world population frequency were cultured for 7 days. At the end of the assay, the proliferation of CFSE-labeled CD4+T cells was analyzed by FACS. The percentage of donors showing CD4+T cells that proliferated was analyzed as a read-out of human immunogenicity risk. As summarized in Table 11, the assay results show that DG to DA mutants (PI-055660) have significantly lower immunogenicity (IMG) risk (18% donor response positive) compared to other DG mutants (36-54% positive response).

Table 11 - Cell binding kinetics of DG variants

A summary of the characteristics of DA variant AdxDC DAR1 ("GPC3_AdxDC DA variant-DAR1" or "DA variant AdxDCDAR1") is listed in Table 12:

Table 12: Characteristics of DA variant AdxDC DAR1

*Measured on unconjugated protein

Example 15: FITC-labeled and PEGylated anti-GPC3 Adnectin

FITC labeling: ADX_6077_F02 and non-binding control Adnectin were reduced with DTT or TCEP and then subjected to G25 gel filtration chromatography or dialysis. An excess of fluorescein-5-maleimide reagent (ThermoScientific) was then added and the mixture was incubated at room temperature for approximately 2 hours followed by G25 gel filtration chromatography or extensive dialysis (3-4 buffer changes). The resulting degree of labeling was measured by absorbance according to the manufacturer's instructions and/or mass spectrometry. The binding affinity of FITC-labeled and PEGylated GPC3 to human and mouse GPC3 was evaluated as described in the previous examples and the results are summarized in Table 13.

Table 13 - Kinetics of Modified GPC3 Adnectins

The data demonstrate that both FITC-labeled and PEGylated anti-GPC3 adnectins retain binding affinity to both human and murine GPC3.

Example 16: Additional features of GPC3_AdxDC DA and DG molecules

In accelerated stability studies, GPC3_AdxDC DA variant-DAR1 showed chemical and biophysical stability at pH 6.0. In addition, its affinity for human GPC3 (by SPR) did not change after 4 weeks at 40°C.

The aspartate isomerization of the DA variants was approximately 4-fold lower than that of the parent DG molecule. After 3 weeks of incubation at 40°C, pH 6 or pH 7, the D80 isomerization percentages of the DG molecules were 3.6 and 2.4, respectively.

GPC3_AdxDC(DG) showed a favorable toxicity profile with weekly dosing (Q7Dx4) in CDF rats (Table 14). No adverse effects were observed in hematology or serum chemistry profiles with weekly dosing (Q7Dx4) of GPC3 AdxDC in CDF rats.

Table 14: Toxicity characteristics of GPC3_AdxDC(DG)

*"NBC" refers to non-binding AdxDC (Adnectin drug conjugate)

Example 17: GPC3 Adnectin Drug Conjugate Binds to Human GPC3 Xenograft Tissue in Vivo

Human GPC3 high-expressing Hep3B xenograft tissue was incubated with FITC-conjugated GPC3-binding Adnectin DG molecule ("GPC3_AdxDC(DG)") DAR1 at a concentration of 0.04 μg/ml or with 0.2 μg/ml non-GPC3-binding Adnectin. The results showed that the GPC3_AdxDC(DG) molecule bound to the Hep3B xenograft tissue, while the non-binding Adnectin did not bind significantly.

Binding to other tissues was also tested and the results showed that GPC3_AdxDC(DG) had some non-specific binding to the placenta. However, the molecule did not significantly bind to stomach, heart, kidney, liver, skin or tonsil tissues.

GPC3_AdxDC(DA)DAR1 showed similar but weaker binding in Hep3B xenografts. Saturation binding was achieved at 0.2 μg/ml.

Example 18: GPC3_AdxDC(DA) is highly effective in xenografts derived from cell lines that highly express Glypican 3

Hep3B (hepatocellular carcinoma; 260,000 GPC3 molecules/cell) xenografts were used in NSG mice. GPC3_AdxDC(DA)DAR1 or non-binding control Adnectin was administered intravenously 3 times per week at the doses indicated in Table 15.

Table 15: Dosages and tumor growth inhibition of Hep3B xenografts in NSG mice

TGI _D26 *: Tumor growth inhibition on day 26

The results shown in Table 15 and Figure 17 indicate that GPC3_AdxDC(DA) is effective in inhibiting Hep3B tumor growth in vivo.

Similar experiments were performed with xenografts derived from a cell line (H446) that underexpresses Glypican 3. H446 cells are small cell lung cancer cells with approximately 40,000 human PC3 molecules/cell. The cells were injected into CB17 SCID mice.

Table 16: Dosages and tumor growth inhibition of H446 cells in CB17 SCID mice

The results shown in Table 16 and Figure 18 indicate that GPC3_AdxDC(DA) slowed the growth of these tumors.

Example 19: Hep3B tumors preferentially take up GPC3_AdxDC relative to normal tissues

Mice were dosed with 0.015 or 0.22 μmol/kg of ³ H-labeled GPC3_AdxDC, and radioactivity was measured by total body autoradiography (QWBA) after 0.17 h, 1 h, 5 h, and 168 h.

The results are shown in Figures 19 and 20, indicating the following:

● Rapid distribution to tumors and highly perfused tissues;

High levels of radioactivity were still present in tumors 168 hours after administration, but no or low levels of radioactivity were present in other tissues;

● Significant radioactivity in the kidneys: approximately 30% of the radioactivity is excreted in the urine; and

●Similar expression patterns between high-dose and low-dose groups.

In similar experiments, mice were dosed with 0.22 μmol/kg of ³ H-labeled GPC3_AdxDC or non-binding AdxDC control, and radioactivity was measured by QWBA after 0.17 h, 1 h, 5 h, and 168 h.

The results shown in Figures 21 and 22 indicate a higher uptake in Hep3B tumors using GPC3_AdxDCs relative to the non-binding control (RGE AdxDCs). The distribution profiles of GPC3_AdxDCs and the non-binding AdxDC control in other tissues were comparable.

The total radioactivity concentration of GPC3_AdxDC in tumors and tissues is provided in Figure 23. The figure shows that the level of GPC3_AdxDC in tumors is much higher than that in other tissues (except kidney).

Example 20: Positional scanning of anti-GPC3 Adnectin

This example describes a positional scan of 6077_F02 in which EIDKPSQ (SEQ ID NO: 369) was removed and PC was added, wherein amino acid 79 (ie, the "D" of "DG") is either G (as in the original clone) or A.

During library construction, these two proteins, which differ only at amino acid position 79, were mixed. Binding to human Glypican 3-biotin was determined at 100 nM, 10 nM, and 1 nM. For each batch, the 10 nM selection elution was compared to the flag elution, and the 1 nM selection elution was also compared to the flag elution. This generated 4 heat maps for each loop: 10 nM when 79 was G; 1 nM when 79 was G; 10 nM when 79 was A and 1 nM when 79 was A.

For the FG loop, the three segments were merged together to show the complete heatmap. For position 79, one heatmap was generated when it was normalized to G, and one heatmap was generated when it was normalized to A.

The results are shown in Figures 24-31 in the form of heat maps. In the heat maps, numbers > 1 indicate favorable substitutions, however, any number > 0.2 is also acceptable as a substitution. The higher the number, the more favorable the substitution. For example, for the DG parent adnectin, the heat map indicates the following:

- In the BC loop (i.e., sequence SDDYHAH (amino acids 15-21 of SEQ ID NO:98)):

o S23 can be substituted by any amino acid;

o D24 is preferably not substituted with any other amino acid, although S and E may be acceptable.

o Other acceptable substitutions can be deduced from the heat map, where any substitution with a number > 0.2 is acceptable, and numbers > 1 are preferred.

Equivalent form

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Table 1 - Sequence Summary

Claims (82)

1. A tenth domain of human fibronectin type III comprising BC, DE and FG loops ¹⁰ Fn 3), wherein said polypeptide has a K of 1 μm or less _D Specific for human glypican 3 (GPC 3)Is combined with sex, and wherein:

(a) The polypeptide comprises BC, DE and FG loops shown in the amino acid sequences of SEQ ID NO 99, 100 and 156 respectively;

(b) The polypeptide comprises BC, DE and FG loops shown in the amino acid sequences of SEQ ID NO 99, 100 and 183 respectively;

(c) The polypeptide comprises BC, DE and FG loops shown in the amino acid sequences of SEQ ID NO 99, 100 and 210 respectively;

(d) The polypeptide comprises BC, DE and FG loops as shown in the amino acid sequences of SEQ ID NO 99, 100 and 237 respectively;

(e) The polypeptide comprises BC, DE and FG loops shown in the amino acid sequences of SEQ ID NO 99, 100 and 264 respectively;

(f) The polypeptide comprises BC, DE and FG loops shown in the amino acid sequences of SEQ ID NO 99, 100 and 291 respectively;

(g) The polypeptide comprises BC, DE and FG loops shown in the amino acid sequences of SEQ ID NO 99, 100 and 318 respectively; or (b)

(h) The polypeptide comprises BC, DE and FG loops as shown in the amino acid sequences of SEQ ID NO 99, 100 and 129, respectively.

2. The polypeptide of claim 1, wherein the ¹⁰ The Fn3 domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 128, 155, 182, 209, 236, 263, 290 and 317.

3. The polypeptide of claim 1 or claim 2, wherein the ¹⁰ The Fn3 domain comprises or consists of the amino acid sequence of SEQ ID No. 263.

4. The polypeptide of claim 1 or claim 2, wherein the ¹⁰ The Fn3 domain comprises or consists of the amino acid sequence of SEQ ID No. 265.

5. The polypeptide of claim 1 or claim 2, wherein the ¹⁰ The Fn3 domain comprises or consists of the amino acid sequence of SEQ ID No. 266.

6. The polypeptide of claim 1 or claim 2, wherein the ¹⁰ Fn3 domain comprises or consists of the amino acid sequence of SEQ ID NO. 267.

7. The polypeptide of claim 1 or claim 2, wherein the ¹⁰ The Fn3 domain comprises or consists of the amino acid sequence of SEQ ID No. 268.

8. The polypeptide of claim 1 or claim 2, wherein the ¹⁰ The Fn3 domain comprises or consists of the amino acid sequence of SEQ ID NO: 269.

9. The polypeptide of claim 1 or claim 2, wherein the ¹⁰ The Fn3 domain comprises or consists of the amino acid sequence of SEQ ID NO. 270.

10. The polypeptide of claim 1 or claim 2, wherein the ¹⁰ The Fn3 domain comprises or consists of the amino acid sequence of SEQ ID NO: 271.

11. The polypeptide of claim 1 or claim 2, wherein the ¹⁰ The Fn3 domain comprises or consists of the amino acid sequence of SEQ ID No. 272.

12. The polypeptide of claim 1 or claim 2, wherein the ¹⁰ The Fn3 domain comprises or consists of the amino acid sequence of SEQ ID No. 273.

13. The polypeptide of claim 1 or claim 2, wherein the ¹⁰ The Fn3 domain comprises or consists of the amino acid sequence of SEQ ID No. 274.

14. The polypeptide of claim 1 or claim 2, wherein the ¹⁰ The Fn3 domain comprises or consists of the amino acid sequence of SEQ ID No. 275.

15. The polypeptide of claim 1 or claim 2, wherein the ¹⁰ The Fn3 domain comprises or consists of the amino acid sequence of SEQ ID NO: 276.

16. The polypeptide of claim 1 or claim 2, wherein the ¹⁰ Fn3 domain comprises or consists of the amino acid sequence of SEQ ID NO 277.

17. The polypeptide of claim 1 or claim 2, wherein the ¹⁰ The Fn3 domain comprises or consists of the amino acid sequence of SEQ ID No. 278.

18. The polypeptide of claim 1 or claim 2, wherein the ¹⁰ The Fn3 domain comprises or consists of the amino acid sequence of SEQ ID No. 279.

19. The polypeptide of claim 1 or claim 2, wherein the ¹⁰ The Fn3 domain comprises or consists of the amino acid sequence of SEQ ID NO. 280.

20. The polypeptide of claim 1 or claim 2, wherein the ¹⁰ Fn3 domain comprises or consists of the amino acid sequence of SEQ ID NO: 281.

21. The polypeptide of claim 1 or claim 2, wherein the ¹⁰ The Fn3 domain comprises or consists of the amino acid sequence of SEQ ID No. 282.

22. The polypeptide of claim 1 or claim 2, wherein the ¹⁰ The Fn3 domain comprises or consists of the amino acid sequence of SEQ ID NO: 283.

23. The polypeptide of claim 1 or claim 2, wherein the ¹⁰ The Fn3 domain comprises or consists of the amino acid sequence of SEQ ID No. 284.

24. The polypeptide of claim 1 or claim 2, wherein the ¹⁰ The Fn3 domain comprises or consists of the amino acid sequence of SEQ ID NO: 285.

25. The polypeptide of claim 1 or claim 2, wherein the ¹⁰ The Fn3 domain comprises or consists of the amino acid sequence of SEQ ID NO. 286.

26. The polypeptide of claim 1 or claim 2, wherein the ¹⁰ Fn3 domain comprises or consists of the amino acid sequence of SEQ ID NO: 287.

27. The polypeptide of claim 1 or claim 2, wherein the ¹⁰ The Fn3 domain comprises or consists of the amino acid sequence of SEQ ID No. 288.

28. The polypeptide of claim 1 or claim 2, wherein the ¹⁰ The Fn3 domain comprises or consists of the amino acid sequence of SEQ ID NO: 289.

29. The polypeptide of any one of the preceding claims, further comprising a heterologous protein.

30. The polypeptide of any one of the preceding claims, further comprising one or more Pharmacokinetic (PK) modules selected from the group consisting of: polyethylene glycol, sialic acid, fc fragment, transferrin, serum albumin binding protein, and serum immunoglobulin binding protein.

31. The polypeptide of any one of the preceding claims, wherein the ¹⁰ The C-terminus of Fn3 domain and the amino acid sequence P _m X _n A modular connection consisting of, wherein P is proline, X is any amino acid, m is an integer of at least 1, and n is 0 or an integer of at least 1.

32. The polypeptide of claim 31, wherein m is 1 or 2 and n is an integer from 1 to 10.

33. The polypeptide of claim 31 or 32, wherein the moiety comprises a cysteine, and the cysteine is linked to the polypeptide ¹⁰ The C-terminus of Fn3 domain.

34. The polypeptide of claim 33, wherein the module consists of the amino acid sequence P _m CX _n Composition, wherein C is cysteine and each X is independently any amino acid.

35. The polypeptide of claim 34, wherein the moiety consists of the amino acid sequence PmCXn ₁ CXn ₂ Composition, wherein each X is independently any amino acid, n ₁ And n ₂ Independently 0 or an integer of at least 1.

36. The polypeptide of claim 35, wherein n ₁ And n ₂ Independently an integer from 1 to 5.

37. The polypeptide of claim 31, wherein the module is selected from the group consisting of: PI, PC, PCC, PID, PIE, PIDK (SEQ ID NO: 382), PIEK (SEQ ID NO: 383), PIDKP (SEQ ID NO: 384), PIEKP (SEQ ID NO: 385), PIDKPS (SEQ ID NO: 386), PIEKPS (SEQ ID NO: 387), PIDKPC (SEQ ID NO: 388), PIEKPC (SEQ ID NO: 389), PIDKPSQ (SEQ ID NO: 390), PIEKPSQ (SEQ ID NO: 391), PIDKPCQ (SEQ ID NO: 392), PIEKPCQ (SEQ ID NO: 393), PHHHHHH (SEQ ID NO: 394), PCHHHHHH (SEQ ID NO: 395), and PCPPPPPHHHHHH (SEQ ID NO: 424).

38. The polypeptide of claim 37, wherein the moiety is PC or PPC.

39. The polypeptide of claim 35, wherein the module is selected from the group consisting of: PCGC (SEQ ID NO: 412), PCPC (SEQ ID NO: 413), PCGSGC (SEQ ID NO: 414), PCPPPC (SEQ ID NO: 415), PCPPPC (SEQ ID NO: 416), PCGSGSGC (SEQ ID NO: 417), PCHHHHHC (SEQ ID NO: 418), PCCHHHHHH (SEQ ID NO: 419), PCGCHHHHHH (SEQ ID NO: 420), PCPCHHHHHH (SEQ ID NO: 421), PCGSGCHHHHHH (SEQ ID NO: 422), PCPPPCHHHHHH (SEQ ID NO: 423) and PCGSGSGCHHHHHH (SEQ ID NO: 425).

40. The polypeptide of claim 39, wherein the moiety is PCPPPC (SEQ ID NO: 416).

41. A polypeptide conjugate comprising the polypeptide of any one of claims 33-40, wherein the cysteine in the C-terminal moiety is conjugated to a heterologous moiety.

42. The polypeptide conjugate of claim 41 wherein the heterologous moiety is a detectable moiety.

43. The polypeptide conjugate of claim 41 wherein the heterologous moiety is a drug moiety and the polypeptide and drug moiety form an FBS-drug conjugate.

44. The FBS-drug conjugate of claim 43, wherein the drug moiety is attached to the drug moiety by a linker ¹⁰ Fn3 domain conjugation, wherein the linker is a hydrazone, thioether, ester, disulfide, peptidyl linker, or peptide-containing linker.

45. The FBS-drug conjugate of claim 44, wherein the linker is a peptidyl linker.

46. The FBS-drug conjugate of claim 45, wherein the peptide-based linker is Val-Cit, ala-Val, val-Ala-Val, lys-Lys, pro-Val-Gly-Val-Val (SEQ ID NO: 467), ala-Asn-Val, val-Leu-Lys, ala-Ala-Asn, cit-Cit, val-Lys, lys, cit, ser, or Glu.

47. The FBS-drug conjugate of any one of claims 43-46, wherein the drug moiety is a cytotoxic drug.

48. The FBS-drug conjugate of claim 47 wherein the cytotoxic drug is selected from the group consisting of:

(a) Enediynes;

(b) Microtubule lysin;

(c) DNA alkylating agents;

(d) Epothilones;

(e) Auristatin;

(f) Pyrrolo-benzodiazepine (PBD) dimers; and

(g) Maytansinoids.

49. The FBS-drug conjugate of claim 48 wherein the drug moiety is:

50. the FBS-drug conjugate according to any of claims 43-48, wherein the drug moiety is a synthetic tubulysin analogue having the structure of formula (II):

51. the FBS-drug conjugate according to any of claims 43-50, wherein said FBS and drug moiety are conjugated with a linker moiety having the structure of formula (III):

52. The FBS-drug conjugate according to claim 51, wherein the drug-module-linker has the structure of formula (IV):

(IV)

wherein maleimide groups are associated with the said ¹⁰ Sulfhydryl reaction of cysteine of Fn3 domain,

thereby in the drug-module-linker and the described ¹⁰ A thioether linkage is formed between Fn3 domains.

53. The FBS-drug conjugate of any one of claims 44-52, wherein the ¹⁰ The Fn3 domain contains a C-terminal module comprising a cysteine.

54. The FBS-drug conjugate of claim 53 wherein the C-terminal module consists of the amino acid sequence P _m CX _n A composition wherein C is cysteine, each X is independently any amino acid, m is an integer of at least 1, and n is 0 or an integer of at least 1.

55. The FBS-drug conjugate of claim 54 wherein m is 1 or 2 and n is an integer from 1 to 10.

56. The FBS-drug conjugate of claim 53 wherein the C-terminal module consists of the amino acid sequence PmCXn ₁ CXn ₂ Composition, wherein each X is independently any amino acid, n ₁ And n ₂ Independently 0 or an integer of at least 1.

57. The FBS-drug conjugate of claim 56 wherein n ₁ And n ₂ Independently an integer from 1 to 5.

58. The FBS-drug conjugate according to claim 54, wherein the C-terminal module is selected from the group consisting of: PI, PC, PCC, PID, PIE, PIDK (SEQ ID NO: 382), PIEK (SEQ ID NO: 383), PIDKP (SEQ ID NO: 384), PIEKP (SEQ ID NO: 385), PIDKPS (SEQ ID NO: 386), PIEKPS (SEQ ID NO: 387), PIDKPC (SEQ ID NO: 388), PIEKPC (SEQ ID NO: 389), PIDKPSQ (SEQ ID NO: 390), PIEKPSQ (SEQ ID NO: 391), PIDKPCQ (SEQ ID NO: 392), PIEKPCQ (SEQ ID NO: 393), PHHHHHH (SEQ ID NO: 394), PCHHHHHH (SEQ ID NO: 395), and PCPPPPPHHHHHH (SEQ ID NO: 424).

59. The FBS-drug conjugate of claim 54, wherein the C-terminal module is PC or PPC.

60. The FBS-drug conjugate of claim 56 wherein the C-terminal module is selected from the group consisting of: PCGC (SEQ ID NO: 412), PCPC (SEQ ID NO: 413), PCGSGC (SEQ ID NO: 414), PCPPPC (SEQ ID NO: 415), PCPPPC (SEQ ID NO: 416), PCGSGSGC (SEQ ID NO: 417), PCHHHHHC (SEQ ID NO: 418), PCCHHHHHH (SEQ ID NO: 419), PCGCHHHHHH (SEQ ID NO: 420), PCPCHHHHHH (SEQ ID NO: 421), PCGSGCHHHHHH (SEQ ID NO: 422), PCPPPCHHHHHH (SEQ ID NO: 423), PCPPPPPHHHHHH (SEQ ID NO: 424), and PCGSGSGCHHHHHH (SEQ ID NO: 425).

61. The FBS-drug conjugate of claim 60, wherein the moiety is PCPPPC (SEQ ID NO: 16).

62. An FBS-drug conjugate having a structure represented by the formula (I),

wherein m is 1 or 2 and Adx is the polypeptide of any one of claims 31-40, and wherein the sulfur atom attached to Adx is the sulfur atom of the thiol group of the cysteine of the polypeptide.

63. A pharmaceutical composition comprising the polypeptide of any one of claims 1-40 or the polypeptide conjugate of any one of claims 41-43, and a pharmaceutically acceptable carrier.

64. A pharmaceutical composition comprising the FBS-drug conjugate according to any of claims 43-62.

65. The composition of claim 63 or 64, wherein the composition is substantially free of endotoxin.

66. An isolated nucleic acid molecule encoding the polypeptide of any one of claims 1-40.

67. An expression vector comprising a nucleotide sequence encoding the polypeptide of any one of claims 1-40.

68. A cell comprising a nucleic acid encoding the polypeptide of any one of claims 1-40.

69. A method of producing the polypeptide of any one of claims 1-40, comprising culturing the cell of claim 68 under conditions suitable for expression of the polypeptide, and purifying the polypeptide.

70. Use of the polypeptide of any one of claims 1-40, the polypeptide conjugate of any one of claims 41-43, or the FBS-drug conjugate of any one of claims 43-62 for the preparation of a pharmaceutical composition for reducing or inhibiting a cancer that expresses glypican 3, wherein the cancer is hepatocellular carcinoma, melanoma, wilms' tumor, hepatoblastoma, ovarian cancer, or squamous lung carcinoma.

71. A kit comprising the polypeptide, polypeptide conjugate, FBS-drug conjugate or pharmaceutical composition of any of claims 1-65 and instructions for use.

72. Use of a polypeptide according to any one of claims 1 to 40 or a polypeptide conjugate according to any one of claims 41 to 43 for the preparation of a pharmaceutical composition for detecting or measuring the binding of said polypeptide to glypican 3.

73. An FBS-drug conjugate having a structure represented by the formula (I),

wherein m is 1 and Adx is a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs 110-117 and 272-289; and wherein the sulfur atom attached to Adx is the sulfur atom of the thiol group of the C-terminal cysteine of the polypeptide.

74. An FBS-drug conjugate having a structure represented by the formula (I),

wherein m is 2 and Adx comprises the amino acid sequences of SEQ ID NOS 119-126 and 281-288; and wherein the sulfur atom attached to Adx is the sulfur atom of the thiol group of each of the two C-terminal cysteines of Adx.

75. An FBS-drug conjugate having a structure represented by the formula (VI),

(VI)

wherein the sulfur atom attached to cysteine is the sulfur atom of the thiol group of cysteine.

76. An FBS-drug conjugate having a structure represented by the formula (VII),

(VII)

wherein the sulfur atom attached to cysteine is the sulfur atom of the thiol group of cysteine.

77. A pharmaceutical composition comprising the FBS-drug conjugate according to any of claims 73-76.

78. Use of the FBS-drug conjugate according to any of claims 73-76 for the manufacture of a pharmaceutical composition for reducing or inhibiting glypican 3-expressing cancer in a subject, wherein said cancer is hepatocellular carcinoma, melanoma, wilms' tumor, hepatoblastoma, ovarian cancer or squamous lung cancer.

79. A kit comprising the FBS-drug conjugate according to any of claims 76-78 or the pharmaceutical composition according to claim 77, and instructions for use.

80. The polypeptide conjugate of claim 42 wherein the detectable moiety is a radioactive material.

81. The polypeptide conjugate of claim 80 wherein the radioactive material is ¹⁸ F、 ⁶⁰ Cu、 ⁶¹ Cu、 ⁶² Cu、 ⁶⁴ Cu、 ⁸⁶ Y、 ⁸⁹ Zr、 ⁶⁶ Ga、 ⁶⁷ Ga、 ⁶⁸ Ga、 ⁴⁴ Sc、 ⁴⁷ Sc、 ¹¹ C、 ¹¹¹ In、 ^114m In、 ¹¹⁴ In、 ¹²⁵ I、 ¹²⁴ I、 ¹³¹ I、 ¹²³ I、 ³² Cl、 ³³ Cl、 ³⁴ Cl、 ⁷⁴ Br、 ⁷⁵ Br、 ⁷⁶ Br、 ⁷⁷ Br、 ⁷⁸ Br、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ⁹⁰ Y、 ¹⁷⁷ Lu、 ⁹⁹ Tc、 ²¹² Bi、 ²¹³ Bi、 ²¹² Pb、 ²²⁵ Ac. Or (b) ¹⁵³ Sm。

82. Use of a polypeptide conjugate of any one of claims 42, 80 or 81 in the manufacture of a pharmaceutical composition for detecting or measuring the presence of said detectable moiety in a sample.